Treatment of pd-l1-positive melanoma using an anti-pd-1 antibody

ABSTRACT

The invention provides a method of treating a melanoma comprising (i) identifying a patient having a PD-L1 positive melanoma and (ii) administering to the patient an anti-PD-1 antibody or an antigen-binding portion thereof (“an anti-PD-1 antibody monotherapy”). The methods of the invention can extend progression-free survival for over 12 months and/or reduces the tumor size at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the tumor size prior to the administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/430,106 filed on Jun. 3, 2019, which is a continuation of U.S.application Ser. No. 15/141,772 filed Apr. 28, 2016, which claimsbenefit to U.S. Provisional Application No. 62/153,954 filed Apr. 28,2015, each of which is incorporated herein by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web, and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 22, 2022, isnamed 3338_0320004_Seqlisting_ST25.txt and is 3,785 bytes in size.

Throughout this application, various publications are referenced inparentheses by author name and date, or by patent No. or patentPublication No. The disclosures of these publications are herebyincorporated in their entireties by reference into this application inorder to more fully describe the state of the art as known to thoseskilled therein as of the date of the invention described and claimedherein. However, the citation of a reference herein should not beconstrued as an acknowledgement that such reference is prior art to thepresent invention.

FIELD OF THE INVENTION

This invention relates to a method of treating PD-L1-positive melanomacomprising administering an anti-PD-1 antibody.

BACKGROUND OF THE INVENTION

Human cancers harbor numerous genetic and epigenetic alterations,generating neoantigens potentially recognizable by the immune system(Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system,comprised of T and B lymphocytes, has powerful anti-cancer potential,with a broad capacity and exquisite specificity to respond to diversetumor antigens. Further, the immune system demonstrates considerableplasticity and a memory component. The successful harnessing of allthese attributes of the adaptive immune system would make immunotherapyunique among all cancer treatment modalities.

Recently, several immune checkpoint pathway inhibitors have begun toprovide new immunotherapeutic approaches for treating cancer, includingthe development of an antibody (Ab), ipilimumab (YERVOY®), that binds toand inhibits Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) for the treatmentof patients with advanced melanoma and the development of antibodiessuch as nivolumab and pembrolizumab (formerly lambrolizumab; USANCouncil Statement, (2013) Pembrolizumab: Statement on a nonproprietaryname adopted by the USAN Council (ZZ-165), Nov. 27, 2013) that bindspecifically to the Programmed Death-1 (PD-1) receptor and block theinhibitory PD-1/PD-1 ligand pathway.

The promise of the emerging field of personalized medicine is thatadvances in pharmacogenomics will increasingly be used to tailortherapeutics to defined sub-populations, and ultimately, individualpatients in order to enhance efficacy and minimize adverse effects.Recent successes include, for example, the development of imatinibmesylate (GLEEVEC®), a protein tyrosine kinase inhibitor that inhibitsthe bcr-abl tyrosine kinase, to treat Philadelphia chromosome-positivechronic myelogenous leukemia (CML); crizotinib (XALKORI®) to treat the5% of patients with late-stage non-small cell lung cancers who express amutant anaplastic lymphoma kinase (ALK) gene; and vemurafenib(ZELBORAF®), an inhibitor of mutated B-RAF protein (V600E-BRAF) which isexpressed in around half of melanoma tumors. However, unlike theclinical development of small molecule agents that target discreteactivating mutations found in select cancer populations, a particularchallenge in cancer immunotherapy has been the identification ofmechanism-based predictive biomarkers to enable patient selection andguide on-treatment management.

SUMMARY OF THE INVENTION

The present disclosure provides a method for treating a melanomacomprising (i) identifying a patient having a PD-L1 positive melanomatumor; and (ii) administering to the patient an anti-PD-1 antibody or anantigen-binding portion thereof that binds specifically to a human PD-1(“anti-PD1 antibody monotherapy”), wherein the patient is notadministered a combination of an anti-PD-1 antibody or anantigen-binding portion thereof and an anti-CTLA-4 antibody or anantigen-binding portion thereof that binds specifically to a humanCTLA-4 (“combination therapy”).

The present disclosure also provides a method for treating a melanomacomprising (i) identifying a patient having a PD-L1 positive melanomatumor; (ii) administering to the patient an anti-PD-1 antibodymonotherapy; and (iii) administering a combination therapy to thepatient, wherein the patient does not show efficacy with the anti-PD-1monotherapy.

The present disclosure further provides a method for treating a melanomacomprising administering to a patient afflicted with a melanoma tumor ananti-PD-1 antibody monotherapy, not a combination therapy, wherein thepatient is identified as having a PD-L1 positive melanoma tumor prior tothe administration.

The present disclosure also provides a method for extending aprogression-free survival period for over 12 months in a patientafflicted with a melanoma tumor comprising administering to the patientan anti-PD-1 antibody monotherapy, wherein the patient is identified ashaving a melanoma tumor expressing PD-L1 prior to the administration andwherein the patient demonstrates progression-free survival for over 12months. In some embodiments, the progression-free survival of thepatient is extended after the administration for over about 13 months,about 14 months, about 15 months, about 16 months, about 17 months,about 18 months, about 2 years, about 3 years, about 4 years, about 5years, about 6 years, about 7 years, about 8 years, about 9 years, orabout 10 years. In one particular embodiment, the progression-freesurvival of the patient is extended for over 14 months.

The present disclosure also provides a method for reducing a tumor sizeat least by 10% in a patient afflicted with a melanoma tumor comprisingadministering to the patient an anti-PD-1 antibody monotherapy, whereinthe patient is identified as having a melanoma tumor expressing PD-L1prior to the administration and wherein the administration reduces thetumor size at least about 10%, about 20%, about 30%, about 40%, or about50% compared to the tumor size prior to the administration.

In certain embodiments, the present disclosure provides a method forselecting a patient suitable for an anti-PD-1 antibody monotherapycomprising (i) identifying a patient having a PD-L1 positive melanomatumor; and (ii) instructing a healthcare provider to administer to thepatient an anti-PD-1 antibody monotherapy.

In some embodiments, the methods disclosed herein further comprisemeasuring a PD-L1 expression on a melanoma tumor. In certainembodiments, the measuring comprises assessing the proportion of cellsin the test tissue sample that express PD-L1 on the cell surface. In oneparticular embodiment, the presence of PD-L1 is determined using anautomated IHC assay.

The present disclosure also provides a kit for treating a patientafflicted with a melanoma tumor, the kit comprising (a) a dosage rangingfrom 0.1 to 10 mg/kg body weight of an anti-PD-1 antibody or anantigen-binding portion thereof; and (b) instructions for using theanti-PD-1 antibody or the antigen-binding portion thereof in a methoddisclosed herein. In certain embodiments, the kit further comprises adosage ranging from 0.1 to 10 mg/kg body weight of an anti-CTLA-4antibody or an antigen-binding portion thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a patient flow diagram of the randomized, double-blind,multicenter, phase 3 trial.

FIGS. 2A-C show progression-free survival data in the intention-to-treatpopulation (FIG. 2A), in patients with positive PD-L1 status (FIG. 2B),and Negative PD-L1 Status (FIG. 2C). Each graph shows the progressionfree survival for patients treated with nivolumab alone (solid line),ipilimumab alone (dashed line), or the combination of nivolumab andipilimumab (dotted line) in months (FIGS. 2A-C). The number at risk inmonths for each of nivolumab, nivolumab plus ipilimumab, and ipilimumabis shown below each x-axis (FIGS. 2A-C). PD-L1 expression status isbased on verified PD-L1 assay data (FIGS. 2B-C).

FIGS. 3A and 3B show subgroup analyses of progression-free survivalamong patients treated with nivolumab alone compared to ipilimumab alone(FIG. 3A) and patients treated with nivolumab plus ipilimumab comparedto ipilimumab alone (FIG. 3B).

FIGS. 4A-4C show the tumor burden change in target lesions in patientstreated with nivolumab alone (FIG. 4A), nivolumab plus ipilimumab (FIG.4B), and ipilimumab alone (FIG. 4C). In each graph, the y-axis shows thebest reduction from baseline in target lesions (%) and the x-axisrepresents each patient (FIGS. 4A-C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to identifying an optimal strategy fortreating a PD-L1-positive melanoma. In particular, the present inventionshows that for a PD-L1-positive melanoma, a monotherapy of an anti-PD-1antibody or an antigen-binding portion thereof is a better option than acombination therapy of an anti-PD-1 antibody or an antigen-bindingportion thereof and an anti-CTLA-4 antibody or an antigen-bindingportion thereof. If the monotherapy of an anti-PD-L1 antibody does notwork in the patient, however, a combination therapy of an anti-PD-1antibody and an anti-CTLA-4 antibody can be used to treat the patient.

Definitions

In order that the present disclosure may be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects of the disclosure, which can be hadby reference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification in its entirety.

“Administering” refers to the physical introduction of a compositioncomprising a therapeutic agent to a subject, using any of the variousmethods and delivery systems known to those skilled in the art. Routesof administration for the formulations disclosed herein includeintravenous, intramuscular, subcutaneous, intraperitoneal, spinal orother parenteral routes of administration, for example by injection orinfusion. The phrase “parenteral administration” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intralymphatic,intralesional, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.In some embodiments, the formulation is administered via anon-parenteral route, in some embodiments, orally. Other non-parenteralroutes include a topical, epidermal or mucosal route of administration,for example, intranasally, vaginally, rectally, sublingually ortopically. Administering can also be performed, for example, once, aplurality of times, and/or over one or more extended periods.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended or undesirable sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment. For example, an adverse event may be associated withactivation of the immune system or expansion of immune system cells(e.g., T cells) in response to a treatment. A medical treatment may haveone or more associated AEs and each AE may have the same or differentlevel of severity. Reference to methods capable of “altering adverseevents” means a treatment regime that decreases the incidence and/orseverity of one or more AEs associated with the use of a differenttreatment regime.

An “antibody” (Ab) shall include, without limitation, a glycoproteinimmunoglobulin which binds specifically to an antigen and comprises atleast two heavy (H) chains and two light (L) chains interconnected bydisulfide bonds, or an antigen-binding portion thereof. Each H chaincomprises a heavy chain variable region (abbreviated herein as V_(H))and a heavy chain constant region. The heavy chain constant regioncomprises three constant domains, C_(H1), C_(H2) and C_(H3). Each lightchain comprises a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprises one constant domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) comprises three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

An immunoglobulin may derive from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. IgGsubclasses are also well known to those in the art and include but arenot limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to theantibody class or subclass (e.g., IgM or IgG1) that is encoded by theheavy chain constant region genes. The term “antibody” includes, by wayof example, both naturally occurring and non-naturally occurringantibodies; monoclonal and polyclonal antibodies; chimeric and humanizedantibodies; human or nonhuman antibodies; wholly synthetic antibodies;and single chain antibodies. A nonhuman antibody may be humanized byrecombinant methods to reduce its immunogenicity in man. Where notexpressly stated, and unless the context indicates otherwise, the term“antibody” also includes an antigen-binding fragment or anantigen-binding portion of any of the aforementioned immunoglobulins,and includes a monovalent and a divalent fragment or portion, and asingle chain antibody.

The term “monoclonal antibody” (“mAb”) refers to a non-naturallyoccurring preparation of antibody molecules of single molecularcomposition, i.e., antibody molecules whose primary sequences areessentially identical, and which exhibits a single binding specificityand affinity for a particular epitope. A mAb is an example of anisolated antibody. MAbs may be produced by hybridoma, recombinant,transgenic or other techniques known to those skilled in the art.

A “human” antibody (HuMAb) refers to an antibody having variable regionsin which both the framework and CDR regions are derived from humangermline immunoglobulin sequences. Furthermore, if the antibody containsa constant region, the constant region is also derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody,” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. The terms “human” antibodies and “fully human”antibodies and are used synonymously.

A “humanized antibody” refers to an antibody in which some, most or allof the amino acids outside the CDR domains of a non-human antibody arereplaced with corresponding amino acids derived from humanimmunoglobulins. In one embodiment of a humanized form of an antibody,some, most or all of the amino acids outside the CDR domains have beenreplaced with amino acids from human immunoglobulins, whereas some, mostor all amino acids within one or more CDR regions are unchanged. Smalladditions, deletions, insertions, substitutions or modifications ofamino acids are permissible as long as they do not abrogate the abilityof the antibody to bind to a particular antigen. A “humanized” antibodyretains an antigenic specificity similar to that of the originalantibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody.

An “anti-antigen” antibody refers to an antibody that binds specificallyto the antigen. For example, an anti-PD-1 antibody binds specifically toPD-1 and an anti-CTLA-4 antibody binds specifically to CTLA-4.

An “antigen-binding portion” of an antibody (also called an“antigen-binding fragment”) refers to one or more fragments of anantibody that retain the ability to bind specifically to the antigenbound by the whole antibody.

A “cancer” refers a broad group of various diseases characterized by theuncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth results in the formation of malignant tumors thatinvade neighboring tissues and may also metastasize to distant parts ofthe body through the lymphatic system or bloodstream. A “cancer” or“cancer tissue” can include a tumor.

“Cytotoxic T-Lymphocyte Antigen-4” (CTLA-4) refers to animmunoinhibitory receptor belonging to the CD28 family. CTLA-4 isexpressed exclusively on T cells in vivo, and binds to two ligands, CD80and CD86 (also called B7-1 and B7-2, respectively). The term “CTLA-4” asused herein includes human CTLA-4 (hCTLA-4), variants, isoforms, andspecies homologs of hCTLA-4, and analogs having at least one commonepitope with hCTLA-4. The complete hCTLA-4 sequence can be found underGenBank Accession No. AAB59385.

The term “progression-free survival,” which can be abbreviated as PFS,as used herein refers to the length of time during and after thetreatment of a solid tumor (i.e., melanoma) that a patient lives withthe disease but it does not get worse.

“Dosing interval,” as used herein, means the amount of time that elapsesbetween multiple doses of a formulation disclosed herein beingadministered to a subject. Dosing interval can thus be indicated asranges.

The term “dosing frequency” as used herein refers to the frequency ofadministering doses of a formulation disclosed herein in a given time.Dosing frequency can be indicated as the number of doses per a giventime, e.g., once a week or once in two weeks.

The use of the term “fixed dose” with regard to a composition of theinvention means that two or more different antibodies in a singlecomposition are present in the composition in particular (fixed) ratioswith each other. In some embodiments, the fixed dose is based on theweight (e.g., mg) of the antibodies. In certain embodiments, the fixeddose is based on the concentration (e.g., mg/ml) of the antibodies. Insome embodiments, the ratio is at least about 1:1, about 1:2, about 1:3,about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120,about 1:140, about 1:160, about 1:180, about 1:200, about 200:1, about180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1,about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1,about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1,about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1 mg firstantibody to mg second antibody. For example, the 3:1 ratio of a firstantibody and a second antibody can mean that a vial can contain about240 mg of the first antibody and 80 mg of the second antibody or about 3mg/ml of the first antibody and 1 mg/ml of the second antibody.

The use of the term “flat dose” with regard to the composition of theinvention means a dose that is administered to a patient without regardfor the weight or body surface area (BSA) of the patient. The flat doseis therefore not provided as a mg/kg dose, but rather as an absoluteamount of the agent (e.g., the anti-CTLA-4 antibody and/or anti-PD-1antibody). For example, a 60 kg person and a 100 kg person would receivethe same dose of the composition (e.g., 240 mg of an anti-PD-1 antibodyand 80 mg of an anti-CTLA-4 antibody in a single fixed dosingformulation vial containing both 240 mg of an anti-PD-1 antibody and 80mg of an anti-CTLA-4 antibody (or two fixed dosing formulation vialscontaining 120 mg of an anti-PD-1 antibody and 40 mg of an anti-CTLA-4antibody, etc.)).

The term “weight based dose” as referred to herein means that a dosethat is administered to a patient is calculated based on the weight ofthe patient. For example, when a patient with 60 kg body weight requires3 mg/kg of an anti-PD-1 antibody in combination with 1 mg/kg of ananti-CTLA-4 antibody, one can draw the appropriate amounts of theanti-PD-1 antibody (i.e., 180 mg) and the anti-CTLA-4 antibody (i.e., 60mg) at once from a 3:1 ratio fixed dosing formulation of an anti-PD1antibody and an anti-CTLA-4 antibody.

The term “single antibody composition” as used herein refers to acomposition comprising antibodies that specifically bind to a singletarget, e.g., PD-1. As used herein, the single antibody composition caninclude a mixture of antibodies if the antibodies specifically bind tothe same target, e.g., nivolumab and pembrolizumab. In otherembodiments, a single antibody composition can include only one specificantibodies, e.g., nivolumab alone or pembrolizumab alone, but not amixture thereof.

The term “anti-PD-1 antibody monotherapy” as used herein includes atherapy of an anti-PD-1 antibody without an anti-CTLA-4 antibodytherapy. The anti-PD-1 antibody monotherapy comprises, consistsessentially of, or consists of administering one or more doses of ananti-PD-1 antibody to a patient in need thereof, but does not includeadministering an anti-CTLA-4 antibody. In one embodiment, the anti-PD-1antibody monotherapy comprises administering one or more doses of ananti-PD-1 antibody to a patient in need thereof, but does not includeadministering an anti-CTLA-4 antibody. In another embodiment, theanti-PD-1 antibody monotherapy comprises administering one or more dosesof an anti-PD-1 antibody to a patient in need thereof, but does notinclude administering an antibody specifically targeting a protein otherthan PD-1. In other embodiments, the anti-PD-1 antibody monotherapycomprises administering one or more doses of an anti-PD-1 antibody to apatient in need thereof, but does not include administering anotheranti-cancer agent.

An “immune response” refers to the action of a cell of the immune system(for example, T lymphocytes, B lymphocytes, natural killer (NK) cells,macrophages, eosinophils, mast cells, dendritic cells and neutrophils)and soluble macromolecules produced by any of these cells or the liver(including antibodies, cytokines, and complement) that results inselective targeting, binding to, damage to, destruction of, and/orelimination from a vertebrate's body of invading pathogens, cells ortissues infected with pathogens, cancerous or other abnormal cells, or,in cases of autoimmunity or pathological inflammation, normal humancells or tissues.

The term “PD-L1 positive” or “PD-L1 expression positive,” relating tocell surface PD-L1 expression, refers to the proportion of cells in atest tissue sample comprising tumor cells and tumor-infiltratinginflammatory cells above which the sample is scored as expressing cellsurface PD-L1. For cell surface expression assayed byimmunohistochemistry (IHC), e.g., with the mAb 28-8, the PD-L1 positivetumor or PD-L1 expression positive tumor means that at least about0.01%, at least about 0.5%, at least about 1%, at least about 2%, atleast about 3%, at least about 4%, at least about 5%, at least about 6%,at least about 7%, at least about 8%, at least about 9%, at least about10%, at least about 15%, at least about 20%, at least about 25%, or atleast about 30% of the total number of cells express PD-L1. PD-L1positive tumor or PD-L1 expression positive tumor can also be expressedherein as tumor expressing PD-L1. In other embodiments, the PD-L1positive tumor or PD-L1 expression positive tumor means that at leastabout 0.1% to at least about 20% of the total number of cells expressPD-L1. In certain embodiments, the PD-L1 positive tumor or PD-L1expression positive tumor means that at least about 0.1% to at leastabout 10% of the total number of cells express PD-L1. In someembodiments, the PD-L1 positive or PD-L1 expression positive tumor meansthat at least about 1% of the total number of cells express PD-L1 on thecell surface. In other embodiments, the PD-L1 positive or PD-L1expression positive tumor means that at least about 5% of the totalnumber of cells express PD-L1 on the cell surface. In one particularembodiment, PD-L1 positive or PD-L1 expression positive tumor means thatat least about 1%, or in the range of 1-5% of the total number of cellsexpress PD-L1 on the cell surface.

“PD-L1 negative” or “PD-L1 expression negative,” relating to cellsurface PD-L1 expression, refers to the lack of a detectable amount ofcell surface PD-L1. For cell surface expression assayed by IHC, e.g.,with the mAb 28-8, a PD-L1 negative tumor or PD-L1 expression negativetumor means that less than 0.01% of cells express a detectable level ofPD-L1. In some embodiments, a PD-L1 negative tumor or PD-L1 expressionnegative tumor means that zero (0) cells express a detectable level ofPD-L1. In some embodiments, a PD-L1 negative or a PD-L1 expressionnegative tumor is any tumor other than a PD-L1 positive or a PD-L1expression positive tumor.

“Programmed Death-1 (PD-1)” refers to an immunoinhibitory receptorbelonging to the CD28 family. PD-1 is expressed predominantly onpreviously activated T cells in vivo, and binds to two ligands, PD-L1and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1),variants, isoforms, and species homologs of hPD-1, and analogs having atleast one common epitope with hPD-1. The complete hPD-1 sequence can befound under GenBank Accession No. U64863.

“Programmed Death Ligand-1 (PD-L1)” is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that down-regulateT cell activation and cytokine secretion upon binding to PD-1. The term“PD-L1” as used herein includes human PD-L1 (hPD-L1), variants,isoforms, and species homologs of hPD-L1, and analogs having at leastone common epitope with hPD-L1. The complete hPD-L1 sequence can befound under GenBank Accession No. Q9NZQ7.

A “patient” as used herein includes any patient who is afflicted with acancer (e.g., melanoma). The terms “subject” and “patient” are usedinterchangeably herein.

A “therapeutically effective amount” or “therapeutically effectivedosage” of a drug or therapeutic agent is any amount of the drug that,when used alone or in combination with another therapeutic agent,protects a subject against the onset of a disease or promotes diseaseregression evidenced by a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.The ability of a therapeutic agent to promote disease regression can beevaluated using a variety of methods known to the skilled practitioner,such as in human subjects during clinical trials, in animal modelsystems predictive of efficacy in humans, or by assaying the activity ofthe agent in in vitro assays.

“Treatment” or “therapy” of a subject refers to any type of interventionor process performed on, or the administration of an active agent to,the subject with the objective of reversing, alleviating, ameliorating,inhibiting, slowing down or preventing the onset, progression,development, severity or recurrence of a symptom, complication orcondition, or biochemical indicia associated with a disease.

A “tumor-infiltrating inflammatory cell” is any type of cell thattypically participates in an inflammatory response in a subject andwhich infiltrates tumor tissue. Such cells include tumor-infiltratinglymphocytes (TILs), macrophages, monocytes, eosinophils, histiocytes anddendritic cells.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of” refer to a value orcomposition that is within an acceptable error range for the particularvalue or composition as determined by one of ordinary skill in the art,which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg caninclude any number between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mgand 3.6 mg (for 20%). Furthermore, particularly with respect tobiological systems or processes, the terms can mean up to an order ofmagnitude or up to 5-fold of a value. When particular values orcompositions are provided in the application and claims, unlessotherwise stated, the meaning of “about” or “comprising essentially of”should be assumed to be within an acceptable error range for thatparticular value or composition.

The terms “about once a week,” “once about every week,” “once aboutevery two weeks,” or any other similar dosing interval terms as usedherein means approximate number, and “about once a week” or “once aboutevery week” can include every seven days±two days, i.e., every five daysto every nine days. The dosing frequency of “once a week” thus can beevery five days, every six days, every seven days, every eight days, orevery nine days. “Once about every two weeks” can include every fourteendays±three days, i.e., every eleven days to every seventeen days.Similar approximations apply, for example, to once about every threeweeks, once about every four weeks, once about every five weeks, onceabout every six weeks and once about every twelve weeks. In someembodiments, a dosing interval of once about every six weeks or onceabout every twelve weeks means that the first dose can be administeredany day in the first week, and then the next dose can be administeredany day in the sixth or twelfth week, respectively. In otherembodiments, a dosing interval of once about every six weeks or onceabout every twelve weeks means that the first dose is administered on aparticular day of the first week (e.g., Monday) and then the next doseis administered on the same day of the sixth or twelfth weeks (i.e.,Monday), respectively.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one-tenth and one-hundredth of an integer), unlessotherwise indicated.

Various aspects of the invention are described in further detail in thefollowing subsections.

Methods of the Invention

The present invention is directed to a method for treating aPD-L1-positive melanoma in a subject in need thereof. In the generalmelanoma population (mix of PD-L1-negative tumors and PD-L1-positivetumors), a combination therapy of an anti-PD-L1 antibody and ananti-CTLA-4 antibody appears to provide better progression-free survivalthan a monotherapy of either an anti-PD-1 antibody or an anti-CTLA-4antibody. See FIG. 2A. However, the present invention identifies that ina PD-L1-positive melanoma population, the progression-free survival ofan anti-PD-1 antibody monotherapy is substantially the same as acombination therapy of an anti-PD-1 antibody and an anti-CTLA-4antibody. See FIG. 2B. Therefore, in patients having a PD-L1-positivetumor, a monotherapy of an anti-PD-1 antibody can be as effective as acombination therapy. In order to reduce the toxicity of the combinationtherapy in the patient, the present invention provides identifying apatient having a PD-L1-positive tumor and providing a monotherapy of ananti-PD-1 antibody for the initial treatment option. The patient can beadministered with a combination therapy of an anti-PD-1 antibody and ananti-CTLA-4 antibody if the initial option fails for any reasons.

In one embodiment, the invention includes a method of treating amelanoma comprising: (i) identifying a patient having a PD-L1-positivemelanoma tumor; and (ii) administering to the patient a single antibodycomposition comprising an anti-PD-1 antibody or an antigen-bindingportion thereof that binds specifically to a human PD-1, wherein thepatient is not administered with a combination of an anti-PD-1 antibodyor an antigen-binding portion thereof and an anti-CTLA-4 antibody or anantigen-binding portion thereof that binds specifically to a humanCTLA-4 (“combination therapy”). The invention can also include a methodfor treating a melanoma comprising: (i) identifying a patient having aPD-L1-positive melanoma tumor; (ii) administering to the patient ananti-PD-1 antibody monotherapy; and (iii) administering a combinationtherapy, wherein the patient does not show efficacy with the anti-PD-1antibody monotherapy.

In another embodiment, the invention includes a method for treating amelanoma comprising: (i) identifying a patient having a PD-L1 positivemelanoma tumor; and (ii) administering to the patient: (a) an anti-PD-1antibody or an antigen-binding portion thereof that binds specificallyto a human PD-1 (“anti-PD1 antibody monotherapy”); or (b) a combinationof an anti-PD-1 antibody or an antigen-binding portion thereof thatbinds specifically to a human PD-1 and an anti-CTLA-4 antibody or anantigen-binding portion thereof that binds specifically to a humanCTLA-4 (“combination therapy”). Selection of either an anti-PD-1antibody monotherapy or a combination therapy can be based on thepatient's prior toxicity profile or prior history with either themonotherapy or the combination therapy.

In another embodiment, the invention includes a method for treating amelanoma comprising administering to a patient afflicted with a melanomatumor an anti-PD1 antibody monotherapy, not a combination therapy,wherein the patient is identified as having a PD-L1-positive melanomatumor prior to the administration. The methods of the present inventioncan further include (i) identifying a patient who does not show efficacywith the anti-PD-1 monotherapy; and (ii) administering a combinationtherapy to the patient who does not show efficacy with the anti-PD-1monotherapy. In one particular embodiment, the invention includes amethod for treating a melanoma comprising: (i) identifying a patienthaving a PD-L1 positive melanoma tumor; (ii) administering to thepatient an anti-PD-1 antibody monotherapy; (iii) identifying a patientthat does not show efficacy with the anti-PD-1 monotherapy; and (iv)administering a combination therapy to the patient who does not showefficacy with the anti-PD-1 monotherapy.

In certain embodiments, the invention includes a method for extending aprogression-free survival period for over 12 months in a patientafflicted with a melanoma tumor comprising administering to the patienta monotherapy of an anti-PD-1 antibody or an antigen-binding portionthereof that binds specifically to a human PD-1, wherein the patient isidentified as having a PD-L1-positive melanoma tumor prior to theadministration and wherein the patient demonstrates progression-freesurvival for over 12 months. In other embodiments, the inventionprovides a method for extending a progression-free survival period forover 12 months in a patient afflicted with a melanoma tumor comprising:(i) identifying a patient having a PD-L1-positive melanoma tumor; (ii)administering to the patient an anti-PD-1 monotherapy; and (iii)administering a combination therapy to the patient who does not showefficacy with the anti-PD-1 monotherapy, wherein the patientdemonstrates progression-free survival for over 12 months. According tothe invention, the progression-free survival of the patient can beextended, after the administration, for over about 13 months, about 14months, about 15 months, about 16 months, about 17 months, about 18months, about 2 years, about 3 years, about 4 years, about 5 years,about 6 years, about 7 years, about 8 years, about 9 years, or about 10years. In a particular embodiment, the progression-free survival of thepatient is extended for over 14 months.

In still other embodiments, the invention is directed to a method forreducing a tumor size at least by 10% in a patient afflicted with amelanoma tumor comprising administering to the patient a monotherapy ofan anti-PD-1 antibody or an antigen-binding portion thereof that bindsspecifically to a human PD-1, wherein the patient is identified ashaving a PD-L1-positive melanoma tumor prior to the administration andwherein the administration reduces the tumor size at least about 10%,about 20%, about 30%, about 40%, or about 50% compared to the tumor sizeprior to the administration. In yet other embodiments, the methodcomprises (i) identifying a patient having a PD-L1-positive melanomatumor; and (ii) administering to the patient an anti-PD-1 antibodymonotherapy; and (iii) administering a combination therapy to thepatient who does not show efficacy with the anti-PD-1 monotherapy,wherein the administration reduces the tumor size at least about 10%,about 20%, about 30%, about 40%, or about 50% compared to the tumor sizeprior to the administration. The tumor size, after the administration,can be reduced at least about 60%, 70%, 80%, 90% or 100%. The tumor canbe completely eliminated from the patient's body after theadministration.

The invention can also include a method of preventing a relapse and/orinduce a remission to a patient comprising administering to the patienta monotherapy of an anti-PD-1 antibody or an antigen-binding portionthereof that binds specifically to a human PD-1, wherein the patient isidentified as having a PD-L1-positive melanoma tumor prior to theadministration. In some embodiments, the method of the inventioncomprises (i) identifying a patient having a PD-L1-positive melanomatumor; (ii) administering to the patient an anti-PD-1 antibody or anantigen-binding portion thereof that binds specifically to a human PD-1as a monotherapy, and not a combination of an anti-PD-1 antibody or anantigen-binding portion thereof and an anti-CTLA-4 antibody or anantigen-binding portion thereof.

In certain embodiments, the invention includes a method for increasingan objective response rate to be higher than 55% in a patientpopulation, wherein each patient of the patient population is afflictedwith a melanoma tumor, in a cancer treatment comprising administering tothe patient a monotherapy of an anti-PD-1 antibody or an antigen-bindingportion thereof that binds specifically to a human PD-1, wherein eachpatient is identified as having a PD-L1 positive melanoma tumor prior tothe administration and wherein the objective response rate is higherthan 55%, 60%, 65%, 70%, or 75%. The method can further compriseadministering a combination therapy of an anti-PD-1 antibody or anantigen-binding portion thereof and an anti-CTLA-4 antibody or anantigen-binding portion thereof to those patients who do not showefficacy with the anti-PD-1 monotherapy. In a particular embodiment, themethod further comprises identifying each patient as having aPD-L1-positive melanoma tumor prior to the administration. In otherembodiments, each patient in the methods can further be characterized by(i) extended progression-free survival for over 12 months, (ii) tumorsize reduction at least about 10%, about 20%, about 30%, about 40%, orabout 50% compared to the tumor size prior to the administration, or(iii) both. In some embodiments, the patient population can be at least100 patients having a PD-L1 positive melanoma tumor. In someembodiments, the patient population can be at least 200, 300, 400, 500,600, 700, 800, 900, or 1000 patients having a PD-L1 positive melanomatumor.

In further embodiments, the invention provides a method for selecting apatient suitable for an anti-PD-1 antibody monotherapy comprising: (i)identifying a patient having a PD-L1-positive melanoma tumor; and (ii)instructing a healthcare provider to administer to the patient amonotherapy of an anti-PD-1 antibody or an antigen-binding portionthereof that binds specifically to a human PD-1. The method can furthercomprise administering the anti-PD-1 antibody or an antigen-bindingportion thereof to the patient.

The disclosure also provides a method for treatment of a subjectafflicted with a melanoma, which method comprises: (a) selecting apatient who has a PD-L1-positive tumor, (b) further selecting a patientwho is not suitable for the anti-PD-1 antibody monotherapy, and (c)administering a combination therapy of an anti-PD-1 antibody and ananti-CTLA-4 antibody or a standard-of-care therapeutic, e.g., ananti-CTLA-4 antibody monotherapy, to the selected patient. The furtherselection of a patient who is not suitable for the monotherapy can bepatients who do not respond to the monotherapy or who have too muchtoxicity to the monotherapy. Those patients may show efficacy to thecombination therapy because, not bound by any theory, the combinationtherapy may utilize a different mechanism of action. In addition, thepatients may be able to tolerate the combination therapy, which may havea lower dose of one or both antibodies.

The methods of the invention as a result of the anti-PD-1 monotherapyand/or the combination therapy can treat the melanoma tumor, reduce thetumor size, prevent growth of the tumor, eliminate the tumor from thepatient, prevent a relapse of a tumor, induce a remission in a patient,or any combination thereof. In certain embodiments, the anti-PD-1monotherapy and/or the combination therapy induces a complete response.In other embodiments, the anti-PD-1 monotherapy and/or the combinationtherapy induces a partial response.

Melanoma

Melanoma (MEL) is a malignant tumor of melanocytes, themelanin-producing cells found predominantly in skin. Though less commonthan other skin cancers, it is the most dangerous of skin cancers if notdiagnosed early and causes the majority (75%) of skin cancer deaths. Theincidence of MEL is increasing worldwide in Caucasian populations,especially where peoples with low amounts of skin pigmentation receiveexcessive ultraviolet light exposure from the sun. In Europe, theincidence rate is <10-20 per 100,000 population; in the USA 20-30 per100,000; and in Australia, where the highest incidence is observed,50-60 per 100,000 (Garbe et al., Eur. J. Cancer. 48(15):2375-90 (2012)).MEL accounts for about 5% of all new cases of cancer in the UnitedStates (U.S.), and the incidence continues to rise by almost 3% peryear. This translates to an estimated 76,690 new cases in the U.S. in2013 with 9,480 associated deaths (Siegel et al., CA Cancer J. Clin.63(1):11-30 (2013)).

For in situ (stage 0) or early-stage MEL (Stages I-II), surgicalexcision is the primary treatment. In general, the prognosis isexcellent for patients with localized disease and tumors 1.0 mm or lessin thickness, with 5-year survival rates of more than 90% (NCCNGUIDELINES®, 2013—Melanoma). Where surgical excision is not feasible forin situ melanoma due to comorbidity or cosmetically sensitive tumorlocation, topical imiquimod (INN) and radiotherapy are emerging astreatments, especially for lentigo maligna. Chemotherapeutic agents fortreating MEL include dacarbazine, temozolomide and imatinib for melanomawith a c-KIT mutation, high-dose interleukin-2, and paclitaxel with orwithout carboplatin. However, these treatments have modest success, withresponse rates below 20% in first-line (1L) and second-line (2L)settings.

For patients with localized melanomas more than 1.0 mm in thickness,survival rates range from 50-90%. The likelihood of regional nodalinvolvement increases with increasing tumor thickness. With Stage IIIMEL (clinically positive nodes and/or in-transit disease), 5-yearsurvival rates range from 20-70%. By far the most lethal is Stage IV MELwhere long-term survival in patients with distant metastatic melanoma isless than 10% (NCCN GUIDELINES®, 2013—melanoma).

The types of melanoma that can be treated with the present methodsinclude, but are not limited to, lentigo maligna, lentigo malignamelanoma, superficial spreading melanoma, acral lentiginous melanoma,nucosal melanoma, nodular melanoma, polypoid melanoma, desmoplasticmelanoma, amelanotic melanoma, soft-tissue melanoma, melanoma with smallnevus-like cells, melanoma with features of a Spitz nevus, or uvealmelanoma. The stages of melanoma that can be treated with the presentmethods include, but are not limited to, (i) Stage I/II (invasivemelanoma): T1a characterized by less than 1.0 mm primary tumorthickness, without ulceration, and mitosis <1/mm²; T1b characterized byless than 1.0 mm primary tumor thickness, with ulceration or mitoses≥1/mm²; T2a characterized by 1.01-2.0 mm primary tumor thickness,without ulceration; (ii) Stage II (high risk melanoma): T2bcharacterized by 1.01-2.0 mm primary tumor thickness, with ulceration;T3a characterized by 2.01-4.0 mm primary tumor thickness, withoutulceration; T3b characterized by 2.01-4.0 mm primary tumor thickness,with ulceration; T4a characterized by greater than 4.0 mm primary tumorthickness, without ulceration; or T4b characterized by greater than 4.0mm primary tumor thickness, with ulceration; (iii) Stage III (regionalmetastasis): N1 characterized by single positive lymph node; N2characterized by two to three positive lymph nodes or regionalskin/in-transit metastasis; or N3 characterized by four positive lymphnodes or one lymph node and regional skin/in-transit metastases; and(iv) Stage IV (distant metastasis): M1a characterized by distant skinmetastasis, normal LDH; M1b characterized by Lung metastasis, normalLDH; or M1c characterized by other distant metastasis or any distantmetastasis with elevated LDH. PD-L1-positive tumors that are treatableby the present methods can have PD-L1 expression on the surface of tumorcells or tumor infiltrating inflammatory cells.

Measurement of PD-L1 Expression

In certain embodiments, identifying a patient suitable for a PD-1monotherapy for the present methods includes measuring or assessing aPD-L1 expression on the surface of the melanoma tumor cells or tumorinfiltrating inflammatory cells. The phrases “tumors expressing PD-L1,”“PD-L1 expressing tumor,” “PD-L1 positive tumor,” and “PD-L1 expressionpositive tumor” are used interchangeably herein. The meaning of thephrases is provided elsewhere herein. The methods of measuring orassessing the PD-L1 expression can be achieved by any methodsapplicable.

In order to assess the PD-L1 expression, in one embodiment, a testtissue sample is obtained from the patient who is in need of thetherapy. In another embodiment, the assessment of PD-L1 expression canbe achieved without obtaining a test tissue sample. In some embodiments,selecting a suitable patient includes (i) optionally providing a testtissue sample obtained from a patient with cancer of the tissue, thetest tissue sample comprising tumor cells and/or tumor-infiltratinginflammatory cells; and (ii) assessing the proportion of cells in thetest tissue sample that express PD-L1 on the surface of the cells basedon an assessment that the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface is higher than a predeterminedthreshold level.

In any of the methods comprising the measurement of PD-L1 expression ina test tissue sample, however, it should be understood that the stepcomprising the provision of a test tissue sample obtained from a patientis an optional step. That is, in certain embodiments the method includesthis step, and in other embodiments, this step is not included in themethod. It should also be understood that in certain embodiments the“measuring” or “assessing” step to identify, or determine the number orproportion of, cells in the test tissue sample that express PD-L1 on thecell surface is performed by a transformative method of assaying forPD-L1 expression, for example by performing a reversetranscriptase-polymerase chain reaction (RT-PCR) assay or an IHC assay.In certain other embodiments, no transformative step is involved andPD-L1 expression is assessed by, for example, reviewing a report of testresults from a laboratory. In certain embodiments, the steps of themethods up to, and including, assessing PD-L1 expression provides anintermediate result that may be provided to a physician or otherhealthcare provider for use in selecting a suitable candidate for thecombination therapy of an anti-PD-1 antibody and an anti-CTLA-4antibody. In certain embodiments, the steps that provide theintermediate result is performed by a medical practitioner or someoneacting under the direction of a medical practitioner. In otherembodiments, these steps are performed by an independent laboratory orby an independent person such as a laboratory technician.

In certain embodiments of any of the present methods, the proportion ofcells that express PD-L1 is assessed by performing an assay to determinethe presence of PD-L1 RNA. In further embodiments, the presence of PD-L1RNA is determined by RT-PCR, in situ hybridization or RNase protection.In other embodiments, the proportion of cells that express PD-L1 isassessed by performing an assay to determine the presence of PD-L1polypeptide. In further embodiments, the presence of PD-L1 polypeptideis determined by immunohistochemistry (IHC), enzyme-linked immunosorbentassay (ELISA), in vivo imaging, or flow cytometry. In some embodiments,PD-L1 expression is assayed by IHC. In other embodiments of all of thesemethods, cell surface expression of PD-L1 is assayed using, e.g., IHC orin vivo imaging.

Imaging techniques have provided important tools in cancer research andtreatment. Recent developments in molecular imaging systems, includingpositron emission tomography (PET), single-photon emission computedtomography (SPECT), fluorescence reflectance imaging (FRI),fluorescence-mediated tomography (FMT), bioluminescence imaging (BLI),laser-scanning confocal microscopy (LSCM) and multiphoton microscopy(MPM), will likely herald even greater use of these techniques in cancerresearch. Some of these molecular imaging systems allow clinicians tonot only see where a tumor is located in the body, but also to visualizethe expression and activity of specific molecules, cells, and biologicalprocesses that influence tumor behavior and/or responsiveness totherapeutic drugs (Condeelis and Weissleder, Cold Spring Harb. Perspect.Biol. 2(12):a003848 (2010)). Antibody specificity, coupled with thesensitivity and resolution of PET, makes immunoPET imaging particularlyattractive for monitoring and assaying expression of antigens in tissuesamples (McCabe and Wu, Cancer Biother. Radiopharm. 25(3):253-61 (2010);Olafsen et al., Protein Eng. Des. Sel. 23(4):243-9 (2010)). In certainembodiments of any of the present methods, PD-L1 expression is assayedby immunoPET imaging. In certain embodiments of any of the presentmethods, the proportion of cells in a test tissue sample that expressPD-L1 is assessed by performing an assay to determine the presence ofPD-L1 polypeptide on the surface of cells in the test tissue sample. Incertain embodiments, the test tissue sample is a FFPE tissue sample. Inother embodiments, the presence of PD-L1 polypeptide is determined byIHC assay. In further embodiments, the IHC assay is performed using anautomated process. In some embodiments, the IHC assay is performed usingan anti-PD-L1 mAb to bind to the PD-L1 polypeptide.

Assaying Cell-Surface PD-L1 Expression by Automated IHC

In one embodiment of the present methods, an automated IHC method isused to assay the expression of PD-L1 on the surface of cells in FFPEtissue specimens. This disclosure provides methods for detecting thepresence of human PD-L1 antigen in a test tissue sample, or quantifyingthe level of human PD-L1 antigen or the proportion of cells in thesample that express the antigen, which methods comprise contacting thetest sample, and a negative control sample, with a mAb that specificallybinds to human PD-L1, under conditions that allow for formation of acomplex between the antibody or portion thereof and human PD-L1. Incertain embodiments, the test and control tissue samples are FFPEsamples. The formation of a complex is then detected, wherein adifference in complex formation between the test sample and the negativecontrol sample is indicative of the presence of human PD-L1 antigen inthe sample. Various methods are used to quantify PD-L1 expression.

In a particular embodiment, the automated IHC method comprises: (a)deparaffinizing and rehydrating mounted tissue sections in anautostainer; (b) retrieving antigen using a decloaking chamber and pH 6buffer, heated to 110° C. for 10 min; (c) setting up reagents on anautostainer; and (d) running the autostainer to include steps ofneutralizing endogenous peroxidase in the tissue specimen; blockingnon-specific protein-binding sites on the slides; incubating the slideswith primary Ab; incubating with a postprimary blocking agent;incubating with NovoLink Polymer; adding a chromogen substrate anddeveloping; and counterstaining with hematoxylin.

For assessing PD-L1 expression in tumor tissue samples, a pathologistexamines the number of membrane PD-L1⁺ tumor cells in each field under amicroscope and mentally estimates the percentage of cells that arepositive, then averages them to come to the final percentage. Thedifferent staining intensities are defined as 0/negative, 1+/weak,2+/moderate, and 3+/strong. Typically, percentage values are firstassigned to the 0 and 3+ buckets, and then the intermediate 1+ and 2+intensities are considered. For highly heterogeneous tissues, thespecimen is divided into zones, and each zone is scored separately andthen combined into a single set of percentage values. The percentages ofnegative and positive cells for the different staining intensities aredetermined from each area and a median value is given to each zone. Afinal percentage value is given to the tissue for each stainingintensity category: negative, 1+, 2+, and 3+. The sum of all stainingintensities needs to be 100%.

Staining is also assessed in tumor-infiltrating inflammatory cells suchas macrophages and lymphocytes. In most cases macrophages serve as aninternal positive control since staining is observed in a largeproportion of macrophages. While not required to stain with 3+intensity, an absence of staining of macrophages should be taken intoaccount to rule out any technical failure. Macrophages and lymphocytesare assessed for plasma membrane staining and only recorded for allsamples as being positive or negative for each cell category. Stainingis also characterized according to an outside/inside tumor immune celldesignation. “Inside” means the immune cell is within the tumor tissueand/or on the boundaries of the tumor region without being physicallyintercalated among the tumor cells. “Outside” means that there is nophysical association with the tumor, the immune cells being found in theperiphery associated with connective or any associated adjacent tissue.

In certain embodiments of these scoring methods, the samples are scoredby two pathologists operating independently, and the scores aresubsequently consolidated. In certain other embodiments, theidentification of positive and negative cells is scored usingappropriate software.

A histoscore is used as a more quantitative measure of the IHC data. Thehistoscore is calculated as follows:

Histoscore=[(% tumor×1(low intensity))+(% tumor×2(medium intensity))+(%tumor×3(high intensity)]

To determine the histoscore, the pathologist estimates the percentage ofstained cells in each intensity category within a specimen. Becauseexpression of most biomarkers is heterogeneous the histoscore is a truerrepresentation of the overall expression. The final histoscore range is0 (no expression) to 300 (maximum expression).

An alternative means of quantifying PD-L1 expression in a test tissuesample IHC is to determine the adjusted inflammation score (AIS) scoredefined as the density of inflammation multiplied by the percent PD-L1expression by tumor-infiltrating inflammatory cells (Taube et al., Sci.Transl. Med. 4(127):127ra37 (2012)).

Anti-PD-1 Antibodies

PD-1 is a key immune checkpoint receptor expressed by activated T and Bcells and mediates immunosuppression. PD-1 is a member of the CD28family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA.Two cell surface glycoprotein ligands for PD-1 have been identified,Programmed Death Ligand-1 (PD-L1) and Programmed Death Ligand-2 (PD-L2),that are expressed on antigen-presenting cells as well as many humancancers and have been shown to down regulate T cell activation andcytokine secretion upon binding to PD-1. Inhibition of the PD-1/PD-L1interaction mediates potent antitumor activity in preclinical models.

HuMAbs that bind specifically to PD-1 with high affinity have beendisclosed in U.S. Pat. Nos. 8,008,449 and 8,779,105. Other anti-PD-1mAbs have been described in, for example, U.S. Pat. Nos. 6,808,710,7,488,802, 8,168,757 and 8,354,509, and PCT Publication No. WO2012/145493. Each of the anti-PD-1 HuMAbs disclosed in U.S. Pat. No.8,008,449 has been demonstrated to exhibit one or more of the followingcharacteristics: (a) binds to human PD-1 with a K_(D) of 1×10⁻⁷ M orless, as determined by surface plasmon resonance using a Biacorebiosensor system; (b) does not substantially bind to human CD28, CTLA-4or ICOS; (c) increases T-cell proliferation in a Mixed LymphocyteReaction (MLR) assay; (d) increases interferon-γ production in an MLRassay; (e) increases IL-2 secretion in an MLR assay; (f) binds to humanPD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1and/or PD-L2 to PD-1; (h) stimulates antigen-specific memory responses;(i) stimulates antibody responses; and (j) inhibits tumor cell growth invivo. Anti-PD-1 antibodies useful for the present invention include mAbsthat bind specifically to human PD-1 and exhibit at least one, at leasttwo, at least three, at least four, or at least five of the precedingcharacteristics.

In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (alsoknown as “OPDIVO®”; formerly designated 5C4, BMS-936558, MDX-1106, orONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitorantibody that selectively prevents interaction with PD-1 ligands (PD-L1and PD-L2), thereby blocking the down-regulation of antitumor T-cellfunctions (U.S. Pat. No. 8,008,449; Wang et al., Cancer Immunol Res.2(9):846-56 (2014)). In another embodiment, the anti-PD-1 antibody orfragment thereof cross-competes with nivolumab. In other embodiments,the anti-PD-1 antibody or fragment thereof binds to the same epitope asnivolumab. In certain embodiments, the anti-PD-1 antibody has the sameCDRs as nivolumab.

In another embodiment, the anti-PD-1 antibody or fragment thereofcross-competes with pembrolizumab. In some embodiments, the anti-PD-1antibody or fragment thereof binds to the same epitope as pembrolizumab.In certain embodiments, the anti-PD-1 antibody has the same CDRs aspembrolizumab. In another embodiment, the anti-PD-1 antibody ispembrolizumab. Pembrolizumab (also known as “KEYTRUDA®”, lambrolizumab,and MK-3475) is a humanized monoclonal IgG4 antibody directed againsthuman cell surface receptor PD-1 (programmed death-1 or programmed celldeath-1). Pembrolizumab is described, for example, in U.S. Pat. Nos.8,354,509 and 8,900,587; see also cancer.gov/drugdictionary?cdrid=695789(last accessed: Dec. 14, 2014). Pembrolizumab has been approved by theFDA for the treatment of relapsed or refractory melanoma.

In other embodiments, the anti-PD-1 antibody or fragment thereofcross-competes with MEDI0608. In still other embodiments, the anti-PD-1antibody or fragment thereof binds to the same epitope as MEDI0608. Incertain embodiments, the anti-PD-1 antibody has the same CDRs asMEDI0608. In other embodiments, the anti-PD-1 antibody is MEDI0608(formerly AMP-514), which is a monoclonal antibody. MEDI0608 isdescribed, for example, in U.S. Pat. No. 8,609,089B2 or incancer.gov/drugdictionary?cdrid=756047 (last accessed Dec. 14, 2014).

In certain embodiments, the first antibody is an anti-PD-1 antagonist.One example of the anti-PD-1 antagonist is AMP-224, which is a B7-DC Fcfusion protein. AMP-224 is discussed in U.S. Publ. No. 2013/0017199 orin cancer.gov/publications/dictionaries/cancer-drug?cdrid=700595 (lastaccessed Jul. 8, 2015).

In other embodiments, the anti-PD-1 antibody or fragment thereofcross-competes with BGB-A317. In some embodiments, the anti-PD-1antibody or fragment thereof binds the same epitope as BGB-A317. Incertain embodiments, the anti-PD-1 antibody has the same CDRs asBGB-A317. In certain embodiments, the anti-PD-1 antibody is BGB-A317,which is a humanized monoclonal antibody. BGB-A317 is described in U.S.Publ. No. 2015/0079109.

In some embodiments, the antibody is Pidilizumab (CT-011), which is anantibody previously reported to bind to PD-1 but which is believed tobind to a different target. Pidilizumab is described in U.S. Pat. No.8,686,119 B2 or WO 2013/014668 A1.

Anti-PD-1 antibodies useful for the disclosed compositions also includeisolated antibodies that bind specifically to human PD-1 andcross-compete for binding to human PD-1 with nivolumab (see, e.g., U.S.Pat. Nos. 8,008,449 and 8,779,105; WO 2013/173223). The ability ofantibodies to cross-compete for binding to an antigen indicates thatthese antibodies bind to the same epitope region of the antigen andsterically hinder the binding of other cross-competing antibodies tothat particular epitope region. These cross-competing antibodies areexpected to have functional properties very similar to those ofnivolumab by virtue of their binding to the same epitope region of PD-1.Cross-competing antibodies can be readily identified based on theirability to cross-compete with nivolumab in standard PD-1 binding assayssuch as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO2013/173223).

In certain embodiments, the antibodies that cross-compete for binding tohuman PD-1 with, or bind to the same epitope region of human PD-1 as,nivolumab are mAbs. For administration to human subjects, thesecross-competing antibodies can be chimeric antibodies, or humanized orhuman antibodies. Such chimeric, humanized or human mAbs can be preparedand isolated by methods well known in the art.

Anti-PD-1 antibodies useful for the compositions of the disclosedinvention also include antigen-binding portions of the above antibodies.It has been amply demonstrated that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; and (iv) a Fvfragment consisting of the V_(L) and V_(H) domains of a single arm of anantibody.

Anti-PD-1 antibodies suitable for use in the disclosed compositions areantibodies that bind to PD-1 with high specificity and affinity, blockthe binding of PD-L1 and or PD-L2, and inhibit the immunosuppressiveeffect of the PD-1 signaling pathway. In any of the compositions ormethods disclosed herein, an anti-PD-1 “antibody” includes anantigen-binding portion or fragment that binds to the PD-1 receptor andexhibits the functional properties similar to those of whole antibodiesin inhibiting ligand binding and upregulating the immune system. Incertain embodiments, the anti-PD-1 antibody or antigen-binding portionthereof cross-competes with nivolumab for binding to human PD-1. Inother embodiments, the anti-PD-1 antibody or antigen-binding portionthereof is a chimeric, humanized or human monoclonal antibody or aportion thereof. In certain embodiments, the antibody is a humanizedantibody. In other embodiments, the antibody is a human antibody.Antibodies of an IgG1, IgG2, IgG3 or IgG4 isotype can be used.

In certain embodiments, the anti-PD-1 antibody or antigen-bindingportion thereof comprises a heavy chain constant region which is of ahuman IgG1 or IgG4 isotype. In certain other embodiments, the sequenceof the IgG4 heavy chain constant region of the anti-PD-1 antibody orantigen-binding portion thereof contains an S228P mutation whichreplaces a serine residue in the hinge region with the proline residuenormally found at the corresponding position in IgG1 isotype antibodies.This mutation, which is present in nivolumab, prevents Fab arm exchangewith endogenous IgG4 antibodies, while retaining the low affinity foractivating Fc receptors associated with wild-type IgG4 antibodies (Wanget al., 2014 Cancer Immunol Res. 2(9):846-56). In yet other embodiments,the antibody comprises a light chain constant region which is a humankappa or lambda constant region. In other embodiments, the anti-PD-1antibody or antigen-binding portion thereof is a mAb or anantigen-binding portion thereof. In certain embodiments of any of thetherapeutic methods described herein comprising administration of ananti-PD-1 antibody, the anti-PD-1 antibody is nivolumab. In otherembodiments, the anti-PD-1 antibody is pembrolizumab. In otherembodiments, the anti-PD-1 antibody is chosen from the human antibodies17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 described in U.S. Pat. No. 8,008,449.In still other embodiments, the anti-PD-1 antibody is MEDI0608 (formerlyAMP-514), AMP-224, or BGB-A317.

Because anti-PD-1 and anti-PD-L1 target the same signaling pathway andhave been shown in clinical trials to exhibit similar levels of efficacyin a variety of cancers, including RCC (see Brahmer et al. (2012) N EnglJ Med 366:2455-65; Topalian et al. (2012a) N Engl J Med 366:2443-54; WO2013/173223), an anti-PD-L1 antibody may be substituted for theanti-PD-1 Ab in any of the therapeutic methods disclosed herein. Incertain embodiments, the anti-PD-L1 antibody is BMS-936559 (formerly12A4 or MDX-1105) (see, e.g., U.S. Pat. No. 7,943,743; WO 2013/173223).In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also knownas RG7446) (see, e.g., Herbst et al. (2013) J Clin Oncol 31(suppl):3000.Abstract; U.S. Pat. No. 8,217,149) or MEDI4736 (Khleif (2013) In:Proceedings from the European Cancer Congress 2013; Sep. 27-Oct. 1,2013; Amsterdam, The Netherlands. Abstract 802). In certain embodiments,the antibodies that cross-compete for binding to human PD-L1 with, orbind to the same epitope region of human PD-L1 as the above-referencesPD-L1 antibodies are mAbs. For administration to human subjects, thesecross-competing antibodies can be chimeric antibodies, or can behumanized or human antibodies. Such chimeric, humanized or human mAbscan be prepared and isolated by methods well known in the art.

Anti-PD-L1 Antibodies

In certain embodiments, the present application encompasses use of ananti-PD-L1 antibody as a monotherapy in lieu of anti-PD-1 antibody. Inone embodiment, the anti-PD-L1 antibody inhibits the binding of PD-L1receptor, i.e., PD-1 to its ligand PD-L1.

Anti-PD-L1 antibodies useful for the invention include antibodiesengineered starting from antibodies having one or more of the V_(H)and/or V_(L) sequences disclosed herein, which engineered antibodies canhave altered properties from the starting antibodies. An anti-PD-L1antibody can be engineered by a variety of modifications as describedabove for the engineering of modified anti-PD-1 antibodies of theinvention.

Anti-PD-L1 antibodies of the invention also include isolated antibodiesselected for their ability to bind to PD-L1 in formalin-fixed,paraffin-embedded (FFPE) tissue specimens. The use of FFPE samples isessential for the long-term follow-up analysis of the correlationbetween PD-L1 expression in tumors and disease prognosis or progression.The use of different antibodies to stain PD-L1 in frozen versus FFPEtissues, and the ability of certain antibodies to distinguish membranousand/or cytoplasmic forms of PD-L1, may account for some of the disparatedata reported in the literature correlating PD-L1 expression withdisease prognosis (Hamanishi et al., Proc. Natl. Acad. Sci. USA104(9):3360-3365 (2007); Gadiot et al., Cancer 117(10):2192-2201(2011)). This disclosure provides several rabbit mAbs that bind withhigh affinity specifically to membranous human PD-L1 in FFPE tissuesamples comprising tumor cells and tumor-infiltrating inflammatorycells.

In some embodiments, an anti-PD-L1 antibody useful for the presentmethods includes mAb 28-8 set forth in SEQ ID NOs. 1 and 2,respectively. The sequences of the heavy and light chain CDR domains ofmAb 28-8, as delineated using the Kabat system, are set forth in SEQ IDNOs. 3-8. In other embodiments, an anti-PD-L1 antibody useful for theinvention comprises mAbs 28-1, 28-12, 29-8 and 20-12 or anantigen-binding portion thereof, for example, including Fab, F(ab′)₂ Fd,Fv, and scFv, di-scFv or bi-scFv, and scFv-Fc fragments, diabodies,triabodies, tetrabodies, and isolated CDRs.

Anti-CTLA-4 Antibodies

Anti-CTLA-4 antibodies of the instant invention bind to human CTLA-4 soas to disrupt the interaction of CTLA-4 with a human B7 receptor.Because the interaction of CTLA-4 with B7 transduces a signal leading toinactivation of T-cells bearing the CTLA-4 receptor, disruption of theinteraction effectively induces, enhances or prolongs the activation ofsuch T cells, thereby inducing, enhancing or prolonging an immuneresponse.

HuMAbs that bind specifically to CTLA-4 with high affinity have beendisclosed in U.S. Pat. Nos. 6,984,720 and 7,605,238. Other anti-PD-1mAbs have been described in, for example, U.S. Pat. Nos. 5,977,318,6,051,227, 6,682,736, and 7,034,121. The anti-PD-1 HuMAbs disclosed inU.S. Pat. Nos. 6,984,720 and 7,605,238 have been demonstrated to exhibitone or more of the following characteristics: (a) binds specifically tohuman CTLA-4 with a binding affinity reflected by an equilibriumassociation constant (K_(a)) of at least about 10⁷ M⁻¹, or about 10⁹M⁻¹, or about 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher, as determined by Biacoreanalysis; (b) a kinetic association constant (k_(a)) of at least about10³, about 10⁴, or about 10⁵ m⁻¹ s⁻¹; (c) a kinetic disassociationconstant (k_(d)) of at least about 10³, about 10⁴, or about 10⁵ m⁻¹ s⁻¹;and (d) inhibits the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86).Anti-CTLA-4 antibodies useful for the present invention include mAbsthat bind specifically to human CTLA-4 and exhibit at least one, atleast two, or at least three of the preceding characteristics.

An exemplary clinical anti-CTLA-4 antibody is the human mAb 10D1 (nowknown as ipilimumab and marketed as YERVOY®) as disclosed in U.S. Pat.No. 6,984,720. Ipilimumab is an anti-CTLA-4 antibody for use in themethods disclosed herein. Ipilimumab is a fully human, IgG1 monoclonalantibody that blocks the binding of CTLA-4 to its B7 ligands, therebystimulating T cell activation and improving overall survival (OS) inpatients with advanced melanoma.

Another anti-CTLA-4 antibody useful for the present methods istremelimumab (also known as CP-675,206). Tremelimumab is human IgG2monoclonal anti-CTLA-4 antibody. Tremelimumab is described inWO/2012/122444, U.S. Publ. No. 2012/263677, or WO Publ. No. 2007/113648A2.

Anti-CTLA-4 antibodies useful for the disclosed composition also includeisolated antibodies that bind specifically to human CTLA-4 andcross-compete for binding to human CTLA-4 with ipilimumab ortremelimumab or bind to the same epitope region of human CTLA-4 asipilimumab or tremelimumab. In certain embodiments, the antibodies thatcross-compete for binding to human CTLA-4 with, or bind to the sameepitope region of human CTLA-4 as does ipilimumab or tremelimumab, areantibodies comprising a heavy chain of the human IgG1 isotype. Foradministration to human subjects, these cross-competing antibodies arechimeric antibodies, or humanized or human antibodies. Usefulanti-CTLA-4 antibodies also include antigen-binding portions of theabove antibodies such as Fab, F(ab′)₂, Fd or Fv fragments.

Dosages

The anti-PD-1 antibody can be administered to a suitable patient intherapeutically effective amounts. For example, the anti-PD-1 antibodycan be administered at a dosage ranging from at least about 0.1 to atleast about 20.0 mg/kg body weight. In certain embodiments, theanti-PD-1 antibody is individually administered at a dosage of at leastabout 0.1, at least about 0.3, at least about 0.5, at least about 1, atleast about 3, at least about 5, at least about 10 or at least about 20mg/kg, e.g., at least about 1 to at least about 10 mg/kg, e.g., at leastabout 1 to at least about 3 mg/kg, e.g., at least about 3 mg/kg, e.g.,at least about 1 mg/kg. The anti-PD-1 antibody can be administered at adosing frequency of at least once about every week, at least once aboutevery 2 weeks, at least once about every 3 weeks, or at least once aboutevery 4 weeks, or at least once a month, for up to 6 to up to 72 doses,or for as long as clinical benefit is observed, or until unmanageabletoxicity or disease progression occurs. In some embodiments, theanti-PD-1 antibody is administered at a dosage of about 1 or about 3mg/kg. In certain embodiments, an anti-PD-1 antibody regimen comprisesadministering the anti-PD-1 antibody to the subject at a dosingfrequency of once about every week, once about every 2 weeks, once aboutevery 3 weeks, or once about every 4 weeks, or once a month for 6 to 72doses, or for as long as clinical benefit is observed, or untilunmanageable toxicity or disease progression occurs. In otherembodiments, the anti-PD-1 antibody is administered at a dosage of about1 mg/kg at a dosing frequency of once about every 3 weeks for up to 48doses.

In other embodiments, the anti-PD-1 antibody is administered at thefollowing dosages: (a) 0.1 mg/kg anti-PD-1 antibody; (b) 0.3 mg/kganti-PD-1 antibody; (c) 1 mg/kg anti-PD-1 antibody; (d) 3 mg/kganti-PD-1 antibody; (e) 5 mg/kg anti-PD-1 antibody; (f) 10 mg/kganti-PD-1 antibody; (g) 0.1 mg/kg anti-PD-1 antibody; (h) 0.3 mg/kganti-PD-1 antibody; (i) 1 mg/kg anti-PD-1 antibody; (j) 3 mg/kganti-PD-1 antibody; (k) 5 mg/kg anti-PD-1 antibody; or (l) 10 mg/kganti-PD-1 antibody. In a particular embodiment, the method includesadministration of 1 mg/kg of an anti-PD-1 antibody.

In some embodiments, the dosage of the anti-PD-1 antibody is keptconstant during the induction dosing schedule and the maintenance dosingschedule. In certain embodiments, a regimen comprises: (i) an inductiondosing schedule comprising administration of the anti-PD-1 antibody at adosing frequency of at least once about every 2 weeks, once about every3 weeks, or once about every 4 weeks, or at least once a month, for atleast 2, 4, 6, 8 or 10 doses, followed by administration of theanti-PD-1 antibody at a dosing frequency of at least once about every 2weeks, once about every 3 weeks, or once about every 4 weeks, or atleast once a month, for at least 2, 4, 6, 8 or 12 doses; followed by(ii) a maintenance dosing schedule comprising administration of theanti-PD-1 antibody at a dosing frequency of at least once about every 8weeks, once about every 12 weeks, or once about every 16 weeks, or atleast about once a quarter, for at least 4, 6, 8, 10, 12 or 16 doses, orfor as long as clinical benefit is observed, or until unmanageabletoxicity or disease progression occurs. In one particular embodiment,the dosing schedule comprises administering about 3 mg/kg of ananti-PD-1 antibody every 2 weeks. In another embodiment, the dosingschedule comprises administering about 1 mg/kg of an anti-PD-1 antibodyevery 3 weeks for 4 doses followed by about 3 mg/kg of an anti-PD-1antibody every 2 weeks.

In certain embodiments of this method, the maintenance dosing schedulecomprises combined administration of up to 4, 6, 8, 10, 12 or 16 dosesof the anti-PD-1 antibody. In other embodiments, the concurrent regimencomprises: (i) an induction dosing schedule comprising administration ofthe anti-PD-1 antibody at a dosing frequency of once about every 2weeks, once about every 3 weeks, or once about every 4 weeks, or once amonth, for 2, 4, 6 or 8 doses, followed by administration of theanti-PD-1 antibody at a dosing frequency of once about every 2 weeks,once about every 3 weeks, or once about every 4 weeks, or once a month,for 2, 4, 6, 8 or 12 doses; followed by (ii) a maintenance dosingschedule comprising administration of the anti-PD-1 antibody at a dosingfrequency of once about every 8 weeks, once about every 12 weeks, oronce about every 16 weeks, or about once a quarter, for 4, 6, 8, 10, 12or 16 doses, or for as long as clinical benefit is observed, or untilunmanageable toxicity or disease progression occurs.

In a particular embodiment, the anti-PD-1 antibody is administered atdosages of about 1 mg/kg anti-PD-1 antibody every three weeks.

In certain embodiments of the present methods, the anti-PD-1 antibody isformulated for parenteral administration, e.g., intravenousadministration.

In some embodiments, the therapeutically effective dosage of theanti-PD-1 antibody or antigen-binding portion thereof is a flat dosing(not a bodyweight based dosing) and comprises about 60 mg, about 80 mg,about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg,about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, orabout 300 mg. In other embodiments, the therapeutically effective dosageof the anti-PD-1 antibody or antigen-binding portion thereof comprisesabout 320 mg, 360 mg, 400 mg, 420 mg, 480 mg, 500 mg, 540 mg, 550 mg,600 mg, 620 mg, 650 mg, 680 mg, 700 mg, 720 mg, 780 mg, 800 mg, 840 mg,or 900 mg. In some embodiments, the dose of the anti-PD-1 antibody inthe composition is between about 60 mg and about 300 mg, between about60 mg and about 100 mg, between about 100 mg and about 200 mg, orbetween about 200 mg and about 300 mg. In some embodiments, the amountof the anti-PD-1 antibody in the composition is at least about 80 mg,about 160 mg, or about 240 mg. In some embodiments, the dose of theanti-PD-1 antibody in the composition is at least about 240 mg or atleast about 80 mg.

In other embodiments, the dosages are given every week, every two weeks,every three weeks, every four weeks, every five weeks, every six weeks,every seven weeks, every eight weeks, every nine weeks, or every 10weeks.

In some embodiments, the anti-PD-1 antibody or antigen-binding portionthereof is administered at a dose of about 240 mg. In embodiments, theanti-PD-1 antibody or antigen-binding portion thereof is administered ata dose of about 360 mg. In embodiments, the anti-PD-1 antibody orantigen-binding portion thereof is administered at a dose of about 480mg. In one embodiment, 360 mg of the anti-PD-1 antibody or antigenbinding fragment is administered once every 3 weeks. In anotherembodiment, 480 mg of the anti-PD-1 antibody or antigen binding fragmentis administered once every 4 weeks.

The combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody canbe administered to a suitable patient in therapeutically effectiveamounts. For example, each antibody can be administered at a dosageranging from at least about 0.1 to at least about 20.0 mg/kg bodyweight. In certain embodiments, each of the anti-PD-1 and anti-CTLA-4antibodies is individually administered at a dosage of at least about0.1, at least about 0.3, at least about 0.5, at least about 1, at leastabout 3, at least about 5, at least about 10 or at least about 20 mg/kg,e.g., at least about 1 to at least about 10 mg/kg, e.g., at least about1 to at least about 3 mg/kg, e.g., at least about 3 mg/kg, e.g., atleast about 1 mg/kg. Each of the anti-PD-1 antibody and anti-CTLA-4antibody can be administered at a dosing frequency of at least onceabout every week, at least once about every 2 weeks, at least once aboutevery 3 weeks, or at least once about every 4 weeks, or at least once amonth, for up to 6 to up to 72 doses, or for as long as clinical benefitis observed, or until unmanageable toxicity or disease progressionoccurs. In some embodiments, the anti-PD-1 antibody is administered at adosage of about 1 or about 3 mg/kg. In certain embodiments, thesequenced regimen comprises administering the anti-PD-1 antibody to thesubject at a dosing frequency of once about every week, once about every2 weeks, once about every 3 weeks, or once about every 4 weeks, or oncea month for 6 to 72 doses, or for as long as clinical benefit isobserved, or until unmanageable toxicity or disease progression occurs.In other embodiments, the anti-PD-1 is administered at a dosage of about1 mg/kg at a dosing frequency of once about every 3 weeks for up to 48doses. In some embodiments, the anti-CTLA-4 antibody is administered ata dosage of 1 or 3 mg/kg. In certain embodiments, the sequenced regimencomprises administering the anti-CTLA-4 antibody to the subject at adosing frequency of once about every week, once about every 2 weeks,once about every 3 weeks, or once about every 4 weeks, or once a monthfor 6 to 72 doses, or for as long as clinical benefit is observed, oruntil unmanageable toxicity or disease progression occurs. In otherembodiments, the anti-CTLA-4 antibody is administered at a dosage ofabout 3 mg/kg at a dosing frequency of once about every 3 weeks for upto 48 doses.

In other embodiments, the anti-PD-1 and anti-CTLA-4 antibodies areadministered at the following dosages: (a) 0.1 mg/kg anti-PD-1 antibodyand 3 mg/kg of anti-CTLA-4 antibody; (b) 0.3 mg/kg anti-PD-1 antibodyand 3 mg/kg of anti-CTLA-4 antibody; (c) 1 mg/kg anti-PD-1 antibody and3 mg/kg of anti-CTLA-4 antibody; (d) 3 mg/kg anti-PD-1 A antibody b and3 mg/kg of anti-CTLA-4 antibody; (e) 5 mg/kg anti-PD-1 antibody and 3mg/kg of anti-CTLA-4 antibody; (f) 10 mg/kg anti-PD-1 antibody and 3mg/kg of anti-CTLA-4 antibody; (g) 0.1 mg/kg anti-PD-1 antibody and 1mg/kg of anti-CTLA-4 antibody; (h) 0.3 mg/kg anti-PD-1 antibody and 1mg/kg of anti-CTLA-4 antibody; (i) 1 mg/kg anti-PD-1 antibody and 1mg/kg of anti-CTLA-4 antibody; (j) 3 mg/kg anti-PD-1 antibody and 1mg/kg of anti-CTLA-4 antibody; (k) 5 mg/kg anti-PD-1 antibody and 1mg/kg of anti-CTLA-4 antibody; or (l) 10 mg/kg anti-PD-1 antibody and 1mg/kg of anti-CTLA-4 antibody. In a particular embodiment, the methodsinclude administration of 1 mg/kg of an anti-PD-1 antibody and 3 mg/kgof an anti-CTLA-4 antibody.

In some embodiments, the dosage of each of the anti-PD-1 and anti-CTLA-4antibodies is kept constant during the induction dosing schedule and themaintenance dosing schedule. In certain embodiments, a regimencomprises: (i) an induction dosing schedule comprising combinedadministration of the anti-PD-1 and anti-CTLA-4 antibodies at a dosingfrequency of at least once about every 2 weeks, once about every 3weeks, or once about every 4 weeks, or at least once a month, for atleast 2, 4, 6, 8 or 10 doses, followed by administration of theanti-PD-1 antibody alone at a dosing frequency of at least once aboutevery 2 weeks, once about every 3 weeks, or once about every 4 weeks, orat least once a month, for at least 2, 4, 6, 8 or 12 doses; followed by(ii) a maintenance dosing schedule comprising combined administration ofthe anti-PD-1 and anti-CTLA-4 antibodies at a dosing frequency of atleast once about every 8 weeks, once about every 12 weeks, or once aboutevery 16 weeks, or at least about once a quarter, for at least 4, 6, 8,10, 12 or 16 doses, or for as long as clinical benefit is observed, oruntil unmanageable toxicity or disease progression occurs.

In certain embodiments of this method, the maintenance dosing schedulecomprises combined administration of up to 4, 6, 8, 10, 12 or 16 dosesof the anti-PD-1 and anti-CTLA-4 antibodies. In other embodiments, theconcurrent regimen comprises: (i) an induction dosing schedulecomprising combined administration of the anti-PD-1 and anti-CTLA-4antibodies at a dosing frequency of once about every 2 weeks, once aboutevery 3 weeks, or once about every 4 weeks, or once a month, for 2, 4, 6or 8 doses, followed by administration of the anti-PD-1 antibody aloneat a dosing frequency of once about every 2 weeks, once about every 3weeks, or once about every 4 weeks, or once a month, for 2, 4, 6, 8 or12 doses; followed by (ii) a maintenance dosing schedule comprisingcombined administration of the anti-PD-1 and anti-CTLA-4 antibodies at adosing frequency of once about every 8 weeks, once about every 12 weeks,or once about every 16 weeks, or about once a quarter, for 4, 6, 8, 10,12 or 16 doses, or for as long as clinical benefit is observed, or untilunmanageable toxicity or disease progression occurs.

In a particular embodiment, the anti-PD-1 and anti-CTLA-4 antibodies areadministered at dosages of about 1 mg/kg anti-PD-1 antibody every threeweeks and about 3 mg/kg of anti-CTLA-4 antibody every three weeks.

In certain embodiments of the present methods, the anti-PD-1 andanti-CTLA-4 antibodies are formulated for parenteral administration,e.g., intravenous administration. In certain other embodiments, when theanti-PD-1 and anti-CTLA-4 antibodies are administered in combination,they are administered within about 30 minutes of each other. Eitherantibody may be administered first, that is, in certain embodiments, theanti-PD-1 antibody is administered before the anti-CTLA-4 antibody,whereas in other embodiments, the anti-CTLA-4 antibody is administeredbefore the anti-PD-1 antibody. Typically, each antibody is administeredintravenously over a period of about 60 minutes. In further embodiments,the anti-PD-1 and anti-CTLA-4 antibodies are administered concurrently,either admixed as a single composition in a pharmaceutically acceptableformulation for concurrent administration, or concurrently as separatecompositions with each antibody in a pharmaceutically acceptableformulation.

In some embodiments, the anti-PD-1 antibody and the anti-CTLA-4 antibodyare formulated in a single composition. The ratio of the amount of theanti-PD-1 antibody and the amount of the anti-CTLA-4 antibody in thesingle composition can be 10:1 to 1:10. In other embodiments, the ratioof the amount of the anti-PD-1 antibody and the amount of theanti-CTLA-4 antibody in the single composition is 5:1, 4:1, 3:1, 2:1,1:1, 1:2, 1:3, 1:4, or 1:5. In a particular embodiment, the ratio of theamount of the anti-PD-1 antibody and the amount of the anti-CTLA-4antibody in the single composition is 1:3.

In some embodiments, the composition is administered at a flat doseregardless of the weight of the patient. For example, each of theanti-PD-1 antibody with the anti-CTLA-4 antibody may be administered ata flat dose of 20, 50, 75, 80, 160, 200, 240, 300, 360, 400, 480, 500,750 or 1500 mg, without regard to the patient's weight. In someembodiments the composition is administered at a weight-based dose atany dose disclosed herein. In some embodiments, the amount of theanti-PD-1 antibody and the amount of the anti-CTLA-4 antibodyadministered to the patient at a single dose are identical.

In other embodiments, the therapeutically effective dosage of theanti-CTLA-4 antibody or antigen-binding portion thereof comprises about60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260mg, about 280 mg, or about 300 mg. In some embodiments, the dose of theanti-CTLA-4 antibody in the composition is between about 60 mg and about300 mg, between about 60 mg and about 100 mg, between about 100 mg andabout 200 mg, or between about 200 mg and about 300 mg. In someembodiments, the amount of the anti-CTLA-4 antibody in the compositionis at least about 80 mg, about 160 mg, or about 240 mg. In someembodiments, the dose of the anti-CTLA-4 antibody in the composition isat least about 240 mg or at least about 80 mg.

In some embodiments, a flat dose of an anti-PD-1 antibody is 80 mg, anda flat dose of an anti-CTLA-4 antibody is 240 mg.

In some embodiments, the anti-PD-1 antibody is administered at asubtherapeutic dose, i.e., a dose of the therapeutic agent that issignificantly lower than the usual or FDA-approved dose whenadministered as monotherapy for the treatment of the cancer. Thequantity of the second antibody in the composition is calculated basedon the desired ratio.

In some embodiments, the composition is administered by intravenousinfusion about once per week, about once every 2 weeks, about once every3 weeks, or about once a month. In certain embodiments, the compositionis administered once every 3 weeks. In some embodiments, the infusionoccurs over at least about 10 minutes, about 20 minutes, about 30minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 2hours, about 3 hours, about 4 hours or about 5 hours.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention can be flat or varied so as toobtain an amount of the active ingredient which is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being unduly toxic to the patient.The selected dosage level will depend upon a variety of pharmacokineticfactors including the activity of the particular compositions of thepresent invention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts. A composition of the present invention can be administeredvia one or more routes of administration using one or more of a varietyof methods well known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

Kits

Also within the scope of the present invention are kits, includingpharmaceutical kits, comprising an anti-PD-1 antibody, for therapeuticuses, and diagnostic kits comprising an anti-PD-L1 antibody for assayingmembranous PD-L1 expression as a biomarker for screening patients forthe combination therapy or for predicting the efficacy of the anti-PD-1monotherapy. In some embodiments, the pharmaceutical kits furthercomprise a combination of an anti-PD-1 antibody and an anti-CTLA-4 Ab,for therapeutic uses. Kits typically include a label indicating theintended use of the contents of the kit and instructions for use. Theterm “label” includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit. In certainembodiments of a pharmaceutical kit, the anti-PD-1 antibody isco-packaged with an anti-CTLA-4 antibody in unit dosage form. In certainembodiments of a diagnostic kit, the anti-PD-L1 antibody is co-packagedwith an anti-PD-1 antibody for performing an assay to detect and/orquantify PD-L1 expression. In one particular embodiment, the diagnostickit further comprises an anti-CTLA-4 antibody.

In certain embodiments, the pharmaceutical kit comprises the anti-humanPD-1 HuMAb, nivolumab. In other embodiments, the pharmaceutical kitcomprises the anti-human PD-L1 HuMAb, BMS-936559. In yet otherembodiments, the pharmaceutical kit comprises the anti-human CTLA-4HuMAb, ipilimumab. In certain embodiments, the diagnostic kit comprisesthe rabbit anti-human PD-L1 mAb, 28-8, comprising the V_(H) and V_(L)regions whose amino acid sequences are set forth in SEQ ID NOs. 1 and 2,respectively. In other embodiments, the diagnostic kit comprises themurine anti-human PD-L1 mAb, 5H1 (Dong et al., Nature Med. 8(8):793-800(2002)).

In some embodiments, the invention provides a kit for treating a patientafflicted with a melanoma tumor, the kit comprising:

-   (a) a dosage ranging from at least about 0.1 to at least about 10    mg/kg body weight of an anti-PD-1 antibody or an antigen-binding    portion thereof; and-   (b) instructions for using the anti-PD-1 antibody or the    antigen-binding portion thereof in the methods disclosed herein.

In another embodiment, the invention provides a kit for treating apatient afflicted with a melanoma tumor, the kit comprising:

-   (a) a dosage ranging from at least about 0.1 to at least about 10    mg/kg body weight of an anti-PD-1 antibody or an antigen-binding    portion thereof;-   (b) a dosage ranging from at least about 0.1 to at least about 10    mg/kg body weight of an anti-CTLA-4 antibody or an antigen-binding    portion thereof; and-   (c) instructions for using the anti-PD-1 antibody or the    antigen-binding portion thereof and the anti-CTLA-4 antibody or the    antigen-binding portion thereof in the methods disclosed herein.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allreferences cited throughout this application are expressly incorporatedherein by reference.

EXAMPLES Example 1

A randomized, double-blind, multicenter, phase 3 trial was conducted toevaluate the safety and efficacy of nivolumab alone or nivolumabcombined with ipilimumab in comparison with ipilimumab alone inpreviously untreated metastatic melanoma.

Patients

Eligible patients had histologically confirmed stage III (unresectable)or stage IV melanoma, and no prior systemic treatment for unresectableor metastatic melanoma. Other eligibility criteria included an age of atleast 18 years, an Eastern Cooperative Oncology Group (ECOG) performancestatus score of 0 (indicating no symptoms) or 1 (indicating mildsymptoms), measurable disease by computed tomography or magneticresonance imaging per RECIST v1.1, availability of tissue collected frommetastatic or unresectable tumors for the assessment of PD-L1 status,and known BRAF V600 mutation status (or consent to BRAF V600 mutationtesting per local standards). Key exclusion criteria were presence ofactive brain metastases, ocular melanoma, or autoimmune disease, and anyprior treatment with an anti-PD-1, anti-PD-L1, anti-PD-L2, oranti-CTLA-4 antibody. Patients who required systemic corticosteroidtreatment or other immunosuppressive medications within 14 days of studydrug administration were excluded.

Study Design and Treatment

In the double-blind, phase 3 study, enrolled patients were randomlyassigned in a 1:1:1 ratio to receive 3 mg of nivolumab per kilogram ofbody weight every 2 weeks (plus ipilimumab-matched placebo), or 1 mg ofnivolumab per kilogram every 3 weeks plus 3 mg of ipilimumab perkilogram every 3 weeks for 4 doses (plus nivolumab-matched placebo),followed by 3 mg of nivolumab per kilogram every 2 weeks for cycle 3 andbeyond, or 3 mg of ipilimumab per kilogram every 3 weeks for 4 doses(plus nivolumab-matched placebo).

Both nivolumab and ipilimumab were administered by intravenous infusion.Randomization was stratified according to tumor PD-L1 status (positivevs. negative or indeterminate), BRAF mutation status (V600 mutationpositive vs. wild-type), and American Joint Committee on Cancermetastasis stage (M0, M1a, or M1b vs. M1c). Treatment continued untilRECIST v1.1-defined disease progression, unacceptable toxicity, orwithdrawal of consent. Patients could be treated beyond progressionprovided they had a clinical benefit without clinical deterioration, anddid not have substantial adverse effects, as assessed by theinvestigator.

Progression-free survival and overall survival were co-primary endpoints. Secondary end points included objective response rate, tumorPD-L1 expression as a predictive biomarker for progression-free andoverall survival, and health-related quality of life. Exploratory endpoints include duration of objective response and safety/tolerability ofstudy drug therapy.

Assessments

Patients were assessed for tumor response, according to RECIST v1.1 at12 weeks after randomization and continuing every 6 weeks for 49 weeks,and then every 12 weeks until progression or treatment discontinuation,whichever occurred later. Progression-free survival was defined as thetime between the date of randomization and the first date of documentedprogression or death, whichever occurred first. Patients treated beyondprogression were considered to have progressive disease at the time ofthe initial progression event, as assessed by the investigator,regardless of subsequent tumor responses. Expression of PD-L1 on thesurface of the tumor cells was assessed in a central laboratory byimmunohistochemistry in formalin-fixed, paraffin-embedded tumorspecimens using a rabbit monoclonal anti-human PD-L1 antibody (clone28-8) and an analytically validated automated assay developed by Dako(Carpinteria, Calif.). PD-L1 positivity was defined as at least 5% oftumor cells showing cell surface PD-L1 staining of any intensity in asection containing at least 100 tumor cells that could be evaluated.Indeterminate status was attributed to samples for which tumorcell-surface expression could not be discerned because of melanincontent or strong cytoplasmic staining.

Any patient who received at least one dose of study drug in each of the3 treatment groups was included in the assessment of safety. Theseverity of adverse events was graded according to the National CancerInstitute Common Terminology Criteria for Adverse Events, version4.0.18. Safety assessments were made continuously during the treatmentphase, and up to 100 days after the last dose of study drug.

Statistical Analysis

A study sample size of approximately 915 patients, randomized to the 3treatment arms in a 1:1:1 ratio, was planned. For the comparison ofprogression-free survival, the number of events projected to be observedat a follow-up of at least 9 months provided approximately 83% power todetect an average hazard ratio of 0.71 with a type I error of 0.005(two-sided). Progression-free survival was compared between nivolumabplus ipilimumab and ipilimumab alone, and between nivolumab alone andipilimumab alone with the use of a two-sided log-rank test stratifiedaccording to PD-L1 status, BRAF mutation status, and metastasis stage(as described above). The study was not designed for a formalstatistical comparison between the nivolumab alone and nivolumab plusipilimumab groups. Hazard ratios and corresponding two-sided 99.5%confidence intervals (CIs) were estimated using a Cox proportionalhazards model, with treatment group as a single covariate, stratified bythe above factors. Progression-free survival curves, medians with 95%CIs, and progression-free survival rates at 6, 12, and 18 months with95% CIs were estimated using Kaplan-Meier methodology. Overall survivalwill be analyzed when all patients have a minimum follow-up of 22months.

Results Patients and Treatment

From July 2013 through March 2014, a total of 1296 patients wereenrolled at 137 centers in Australia, Europe, Israel, New Zealand, andNorth America. A total of 945 patients underwent randomization: 316patients were assigned to the nivolumab group, 314 to the nivolumab plusipilimumab group, and 315 to the ipilimumab group (FIG. 1). Baselinecharacteristics were balanced across the three groups. A total of 58.0%had stage M1c disease, 36.1% had an elevated lactate dehydrogenaselevel, 31.5% had a BRAF mutation, and 26.5% had positive PD-L1 status(Table 1).

All randomized patients had been followed for a minimum of 9 months atthe time of database lock (Feb. 17, 2015); 117 of 313 patients (37.4%)in the nivolumab group, 93 of 313 patients (29.7%) in the nivolumab plusipilimumab group, and 50 of 311 patients (16.1%) in the ipilimumab groupwere continuing study treatment (Table 2). The most frequent reason fordiscontinuation was disease progression in the nivolumab and ipilimumabmonotherapy groups (154 of 313 patients [49.2%] and 202 of 311 patients[65.0%], respectively), versus study drug toxicity in the nivolumab plusipilimumab group (120 of 313 patients [38.3%]). The number of patientswho had died was 85 (27.2%) in the nivolumab group, 86 (27.5%) in thenivolumab plus ipilimumab group, and 114 (36.7%) in the ipilimumabgroup.

The median number of doses in patients who received nivolumab alone oripilimumab alone was 15 (range 1-38) and 4 (1-4), respectively. In thecombination group, the median number of doses was 4 (range 1-39) fornivolumab and 4 (range 1-4) for ipilimumab; 147 of 313 patients (47%)received four or more doses of nivolumab monotherapy after combinationtreatment.

TABLE 1 Baseline Characteristics of the Patients. Nivolumab Nivolumabplus Ipilimumab alone Ipilimumab alone Total Characteristic (N = 316) (N= 314) (N = 315) (N = 945) Age—yr Mean 58.7 59.3 60.8 59.6 Range 25-9018-88 18-89 18-90 Age groups—no. (%) <65 yr 198 (62.7) 185 (58.9) 182(57.8) 565 (59.8) ≥65, <75 yr  79 (25.0)  94 (29.9)  89 (28.3) 262(27.7) ≥75 yr  39 (12.3)  35 (11.1)  44 (14.0) 118 (12.5) Sex—no. (%)Male 202 (63.9) 206 (65.6) 202 (64.1) 610 (64.6) Female 114 (36.1) 108(34.4) 113 (35.9) 335 (35.4) ECOG performance status—no. (%) 0 238(75.3) 230 (73.2) 224 (71.1) 692 (73.2) 1  77 (24.4)  83 (26.4)  91(28.9) 251 (26.6) 2  1 (0.3) 0 0  1 (0.1) Not reported 0  1 (0.3) 0  1(0.1) M stage—no. (%) M1c 184 (58.2) 181 (57.6) 183 (58.1) 548 (58.0)M0, M1a, or M1b 132 (41.8) 133 (42.4) 132 (41.9) 397 (42.0) Lactatedehydrogenase—no. (%)* ≤ULN 196 (62.0) 199 (63.4) 194 (61.6) 589(62.3) >ULN 112 (35.4) 114 (36.3) 115 (36.5) 341 (36.1) ≤2 × ULN 271(85.8) 276 (87.9) 279 (88.6) 826 (87.4) >2 × ULN  37 (11.7)  37 (11.8)30 (9.5) 104 (11.0) Unknown  8 (2.5)  1 (0.3)  6 (1.9) 15 (1.6) Brainmetastases at baseline—no. (%) Yes  8 (2.5) 11 (3.5) 15 (4.8) 34 (3.6)No 308 (37.5) 303 (96.5) 300 (95.2) 911 (96.4) PD-L1 status—no. (%)Positive  80 (27.8)  68 (24.5)  75 (27.1) 223 (26.4) Negative 208 (72.2)210 (75.5) 202 (72.9) 620 (73.5) BRAF status—no. (%) Mutation 100 (31.6)101 (32.2)  97 (30.8) 298 (31.5) No mutation 216 (68.4) 213 (67.8) 218(69.2) 647 (68.5) *ULN denotes upper limit of normal.

Efficacy

The median progression-free survival was 6.5 months (95% confidenceinterval [CI], 4.3 to 9.5) in the nivolumab group, 11.5 months (95% CI,8.9 to 16.5) in the nivolumab plus ipilimumab group, and 2.9 months (95%CI, 2.8 to 3.4) in the ipilimumab group (FIG. 2A). A significantimprovement in progression-free survival was observed in the nivolumabplus ipilimumab group as compared with the ipilimumab group (hazardratio, 0.42; 95% CI, 0.31 to 0.57; P<0.0001) (FIG. 2A). A significantimprovement in progression-free survival was also observed in thenivolumab group as compared with the ipilimumab group (hazard ratio,0.57; 95% CI, 0.43 to 0.76; P<0.00001) (FIG. 2A). The hazard ratio forthe comparison between nivolumab plus ipilimumab and nivolumab groupswas 0.74 (95% CI, 0.60 to 0.92).

Analyses of progression-free survival among prespecified patientsubgroups showed a consistent improvement with nivolumab or nivolumabplus ipilimumab as compared with ipilimumab, including subgroups definedby PD-L1 status, BRAF mutation status, and metastasis stage (FIGS. 3Aand 3B). In the combination group, median PFS was 11.7 months (95% CI,8.0 to not reached) among patients with a BRAF mutation and was 11.2months (95% CI, 8.3 to not reached) in patients with wild-type BRAF(FIG. 3B). For patients with a positive PD-L1 tumor status, medianprogression-free survival in the nivolumab, nivolumab plus ipilimumab,and ipilimumab groups was 14.0 months (95% CI, 9.1 to not reached), 14.0months (95% CI, 9.7 to not reached), and 3.9 months (95% CI, 2.8 to4.2), respectively (FIG. 2B). For patients with a negative PD-L1 tumorstatus, median progression-free survival in the nivolumab, nivolumabplus ipilimumab, and ipilimumab groups was 5.3 months (95% CI, 2.8 to7.1 months), 11.2 months (95% CI, 8.0 to not reached) and 2.8 months(95% CI, 2.8 to 3.1), respectively (FIG. 2C).

Investigator-assessed objective response rates were 43.7% (95% CI, 38.1to 49.3%), 57.6% (95% CI, 52.0 to 63.2), and 19.0% (95% CI, 14.9 to23.8) in the nivolumab, nivolumab plus ipilimumab, and ipilimumabgroups, respectively (Table 3). The percentage of patients with acomplete response was higher in the nivolumab plus ipilimumab group thanin either the nivolumab or ipilimumab alone groups (11.5% vs. 8.9% and2.2%) (Table 3). Time to objective response was similar in each group(Table 3), and the median duration of response was not reached in any ofthe groups.

Median reduction in the sum of the longest diameters of tumor targetlesions was −34.5% (interquartile range: −75.4 to 15.4), −51.1% (−75.8to −10.2), and 5.8% (−28.0 to 33.3) in the nivolumab, nivolumab plusipilimumab, and ipilimumab groups, respectively (FIG. 4). Among patientswith PD-L1-positive tumors, the objective response rates were 57.5% (95%CI, 45.9 to 68.5), 72.1% (95% CI, 59.9 to 82.3), and 21.3% (95% CI, 12.7to 32.3) for the nivolumab, nivolumab plus ipilimumab, and ipilimumabgroups, respectively; in patients with PD-L1-negative tumors, theobjective response rates were 41.3% (95% CI, 34.6 to 48.4), 54.8% (95%CI, 47.8 to 61.6), and 17.8% (95% CI, 12.8 to 23.8) (Table 4).

Adverse Events

Treatment-related adverse events of any grade occurred in 82.1%, 95.5%,and 86.2% of patients in the nivolumab, nivolumab plus ipilimumabgroups, and ipilimumab groups, respectively (Table 5). The most commonadverse events in the nivolumab group were fatigue (in 34.2% ofpatients), rash (in 21.7%), and diarrhea (in 19.2%). In the nivolumabplus ipilimumab and ipilimumab groups, diarrhea (in 44.1% and 33.1% ofpatients, respectively), fatigue (in 35.1% and 28.0%), and pruritus (in33.2% and 35.4%) were most common (Table 5). The incidence oftreatment-related adverse events of grade 3 or 4 was also higher in thenivolumab plus ipilimumab group than in either the nivolumab oripilimumab groups (55.0% vs. 16.3% and 27.3%), with diarrhea being themost common (2.2%, 9.3%, and 6.1% in the nivolumab, nivolumab plusipilimumab, and ipilimumab groups, respectively) (Table 5).Treatment-related adverse events of any grade leading to discontinuationoccurred in 7.7%, 36.4%, and 14.8% of patients in the nivolumab,nivolumab plus ipilimumab, and ipilimumab groups, respectively, with themost common being diarrhea (in 1.9%, 8.3%, and 4.5%, respectively) andcolitis (in 0.6%, 8.3%, and 7.7%, respectively) (Table 5). One death dueto study-drug toxicity was reported in the nivolumab group (neutropenia)and one in the ipilimumab group (cardiac arrest), although such adverseevents have not been associated with these drugs in prior studies. Notreatment-related deaths were reported in the combination group.

TABLE 2 Patient Disposition.* Nivolumab Nivolumab plus Ipilimumab aloneIpilimumab alone (N = 313) (N = 313) (N = 311) Patients in the treatmentperiod—no. (%) Continuing 117 (37.4)  93 (29.7) 50 (16.1) Not continuing196 (62.6)  220 (70.3)  261 (83.9)  Reason for not continuing thetreatment—no. (%) Disease progression 154 (49.2)  69 (22.0) 202 (65.0) Study drug toxicity 27 (8.6)  120 (38.3)  47 (15.1) Adverse eventunrelated to 5 (1.6) 12 (3.8)  4 (1.3) study drug Patient request todiscontinue 5 (1.6) 5 (1.6) 4 (1.3) treatment Death 5 (1.6) 5 (1.6) 4(1.3) Maximum clinical benefit 2 (0.6) 2 (0.6) 0 Poor/non-compliance 1(0.3) 1 (0.3) 1 (0.3) Patient withdrew consent 0 3 (1.0) 0 Lost tofollow-up 1 (0.3) 0 0 Patient no longer meets study 0 1 (0.3) 0 criteriaOther 0 3 (1.0) 2 (0.6) Patients in the study—no. (%) Continuing 223(71.2)  221 (70.6)  189 (60.8)  Died 85 (27.2) 86 (27.5) 114 (36.7)  Notcontinuing 5 (1.6) 6 (1.9) 8 (2.6) *At the time of database lock on Feb.17, 2015.

TABLE 3 Response to Treatment. Nivolumab Nivolumab plus Ipilimumab aloneIpilimumab alone Response (N = 316) (N = 314) (N = 315) Best overallresponse—no. (%)^(†) Complete response 28 (8.9) 36 (11.5) 7 (2.2)Partial response 110 (34.8) 145 (46.2)  53 (16.8) Stable disease  34(10.8) 41 (13.1) 69 (21.9) Progressive disease 119 (37.7) 71 (22.6) 154(48.9)  Could not be determined 25 (7.9) 21 (6.7)  32 (10.2) Objectiveresponse^(‡) No. of patients (% [95% CI]) 138 (43.7 [38.1-49.3]) 181(57.6 [52.0-63.2]) 60 (19.0 [14.9-23.8]) Estimated odds ratio (95%CI)^(§) 3.40 (2.02-5.72) 6.11 (3.59-10.38) — Two-sided P value <0.00001<0.00001 — Time to objective response—mo No. of responders 138 181 60Median 2.78 2.76 2.79 Range 2.3-12.5 1.1-11.6 2.5-12.4 ^(†)Best overallresponse were assessed by the investigators with the use of RECIST v1.1.^(‡)Data include patients with a complete response and those with apartial response. ^(§)Relative to ipilimumab alone.

TABLE 4 Objective Response Rate by PD-L1 Status. PD-L1 PositiveNivolumab plus Nivolumab alone Ipilimumab Ipilimumab alone Objectiveresponse^(†) (N = 80) (N = 68) (N = 75) No. of patients (% [95% CI]) 46(57.5 [45.9-68.5]) 49 (72.1 [59.9-82.3]) 16 (21.3 [12.7-32.3]) Estimatedodds ratio (95% CI)^(§) 5.03 (2.44-10.37) 10.41 (4.63-23.40) — PD-L1Negative Nivolumab plus Nivolumab alone Ipilimumab Ipilimumab aloneObjective response^(†) (N = 208) (N = 210) (N = 202) No. of patients (%[95% CI]) 86 (41.3 [34.6-48.4]) 115 (54.8 [47.8-61.6]) 36 (17.8[12.8-23.8]) Estimated odds ratio (95% CI)^(§) 3.25 (2.05-5.13) 5.90(3.71-9.38) — ^(†)Best overall response was assessed by theinvestigators with the use of RECIST v1.1. ^(§)Relative to ipilimumabalone.

The most frequent grade 3 or 4 treatment-related select adverse eventswere diarrhea (2.2%, 9.3%, and 6.1% of patients in the nivolumab,nivolumab plus ipilimumab, and ipilimumab groups, respectively), colitis(in 0.6%, 7.7%, and 8.7%, respectively), increased alanineaminotransferase (in 1.3%, 8.3%, and 1.6%, respectively), and increasedaspartate aminotransferase (in 1.0%, 6.1%, and 0.6%, respectively)(Table 6). With the use of immune modulatory agents, resolution ratesfor grade 3 or 4 select adverse events were generally similar acrosstreatment groups, and were between 85-100% across organ categories inthe nivolumab plus ipilimumab group. As observed in prior studies, mostendocrine events in all treatment groups did not resolve (Table 7).

Discussion

In this randomized, double-blind, phase 3 study, both nivolumab aloneand the combination of nivolumab and ipilimumab significantly increasedprogression-free survival and objective response rates, as compared withipilimumab alone, in previously untreated advanced melanoma. Theseresults were observed independently of PD-L1 tumor status, BRAF mutationstatus, or metastasis stage. Baseline characteristics of studyparticipants were typical of patients with advanced melanoma, althoughthe BRAF mutation rate (31.5%) was lower than the 40-50% generallyreported for advanced disease. While not a primary end point of thestudy, the combination of nivolumab and ipilimumab resulted innumerically longer progression-free survival and a higher response rateas compared with nivolumab alone in the overall study population. Whiletime to response was similar between groups, the first tumor assessmentwas done at week 12 and thus the possibility that responses may haveoccurred earlier with the combination remains unknown.

TABLE 5 Adverse Events (Safety Population).* Nivolumab plus Nivolumabalone Ipilimumab Ipilimumab alone (N = 313) (N = 313) (N = 311) GradeGrade Grade Event Total 3 or 4 Total 3 or 4 Total 3 or 4 no. of patientswith event (%) Any adverse event 311 (99.4) 136 (43.5) 312 (99.7) 215(68.7) 308 (99.0) 173 (55.6) Treatment-related 257 (82.1)  51 (16.3) 299(95.5) 172 (55.0) 268 (86.2)  85 (27.3) adverse event^(†) Diarrhea  60(19.2)  7 (2.2) 138 (44.1)  29 (9.3) 103 (33.1)  19 (6.1) Fatigue 107(34.2)  4 (1.3) 110 (35.1)  13 (4.2)  87 (28.0)  3 (1.0) Pruritus  59(18.8)  0 104 (33.2)  6 (1.9) 110 (35.4)  1 (0.3) Rash  81 (25.9)  2(0.6) 126 (40.3)  15 (4.8) 102 (32.8)  6 (1.9) Nausea  41 (13.1)  0  81(25.9)  7 (2.2)  50 (16.1)  2 (0.6) Pyrexia  18 (5.8)  0  58 (18.5)  2(0.6)  21 (6.8)  1 (0.3) Decreased appetite  34 (10.9)  0  56 (17.9)  4(1.3)  39 (12.5)  1 (0.3) Increase in alanine  12 (3.8)  4 (1.3)  55(17.6)  26 (8.3)  12 (3.9)  5 (1.6) aminotransferase Vomiting  20 (6.4) 1 (0.3)  48 (15.3)  8 (2.6)  23 (7.4)  1 (0.3) Increase in aspartate 12 (3.8)  3 (1.0)  48 (15.3)  19 (6.1)  11 (3.5)  2 (0.6)aminotransferase Hypothyroidism  27 (8.6)  0  47 (15.0)  1 (0.3)  13(4.2)  0 Colitis  4 (1.3)  2 (0.6)  37 (11.8)  24 (7.7)  36 (11.6)  27(8.7) Arthralgia  24 (7.7)  0  33 (10.5)  1 (0.3)  19 (6.1)  0 Headache 23 (7.3)  0  32 (10.2)  1 (0.3)  24 (7.7)  1 (0.3) Dyspnea  14 (4.5)  1(0.3)  32 (10.2)  2 (0.6)  13 (4.2)  0 Treatment-related  24 (7.7)  16(5.1) 114 (36.4)  92 (29.4)  46 (14.8)  41 (13.2) adverse event leadingto discontinuation *The severity of adverse events was graded accordingto the National Cancer Institute Common Terminology Criteria for AdverseEvents, version 4.0. ^(†)The treatment-related adverse events listedhere were reported in at least 10% of the patients in any of the threestudy groups.

TABLE 6 Treatment-related Select Adverse Events (Safety Population).*Nivolumab plus Nivolumab alone Ipilimumab Ipilimumab alone (N = 313) (N= 313) (N = 311) Grade Grade Grade Event Total 3 or 4 Total 3 or 4 Total3 or 4 no. of patients with event (%) Any select adverse 194 (62.0)  24(7.7) 275 (87.9) 124 (39.6) 229 (73.6)  58 (18.6) eventTreatment-related select adverse event^(†) Skin 131 (41.9)  5 (1.6) 185(59.1)  18 (5.8) 168 (54.0)  9 (2.9) Pruritus  59 (18.8)  0 104 (33.2) 6 (1.9) 110 (35.4)  1 (0.3) Rash  68 (21.7)  1 (0.3)  89 (28.4)  9(2.9)  65 (20.9)  5 (1.6) Rash maculo-papular  13 (4.2)  1 (0.3)  37(11.8)  6 (1.9)  37 (11.9)  1 (0.3) Vitiligo  23 (7.3)  1 (0.3)  21(6.7)  0  12 (3.9)  0 Gastrointestinal  61 (19.5)  7 (2.2) 145 (46.3) 46 (14.7) 114 (36.7)  36 (11.6) Diarrhea  60 (19.2)  7 (2.2) 138 (44.1) 29 (9.3) 103 (33.1)  19 (6.1) Colitis  4 (1.3)  2 (0.6)  37 (11.8)  24(7.7)  36 (11.6)  27 (8.7) Hepatic  20 (6.4)  8 (2.6)  94 (30.0)  59(18.8)  22 (7.1)  5 (1.6) Increase in alanine  12 (3.8)  4 (1.3)  55(17.6)  26 (8.3)  12 (3.9)  5 (1.6) aminotransferase Increase inaspartate  12 (3.8)  3 (1.0)  48 (15.3)  19 (6.1)  11 (3.5)  2 (0.6)aminotransferase Endocrine  45 (14.4)  2 (0.6)  94 (30.0)  15 (4.8)  34(10.9)  7 (2.3) Hypothyroidism  27 (8.6)  0  47 (15.0)  1 (0.3)  13(4.2)  0 Hyperthyroidism  13 (4.2)  0  31 (9.9)  3 (1.0)  3 (1.0)  0Hypophysitis  2 (0.6)  1 (0.3)  24 (7.7)  5 (1.6)  12 (3.9)  6 (1.9)Pulmonary  5 (1.6)  1 (0.3)  22 (7.0)  3 (1.0)  6 (1.9)  1 (0.3)Pneumonitis  4 (1.3)  1 (0.3)  20 (6.4)  3 (1.0)  5 (1.6)  1 (0.3) *Theseverity of adverse events was graded according to the National CancerInstitute Common Terminology Criteria for Adverse Events, version 4.0.^(†)The treatment-related select adverse events listed here werereported in at least 5% of the patients in any of the three studygroups.

TABLE 7 Management of Treatment-related Select Adverse Events WithImmune Modulatory Medication (IMM). Nivolumab Nivolumab plus IpilimumabIpilimumab Patients Patients Patients with with with resolution Medianresolution Median resolution Median Select Patients of AE after time toPatients of AE after time to Patients of AE after time to Adversemanaged treatment resolution, managed treatment resolution, managedtreatment resolution, Event Organ with IMM, with IMM, weeks with IMM,with IMM, weeks with IMM, with IMM, weeks Category n (%) n (%) (95% CI)n (%) n (%) (95% CI) n (%) n (%) (95% CI) Skin 33/131 17 (54.8) 34.980/185 (43.2) 55 (75.3) 8.6 (7.0, 12.6) 58/168 (34.5) 41 (74.5) 12.4(25.2) (18.0, NE) (8.9, 19.1) Grade 3-4 3/5 (60.0) 3 (75.0) 2.1 (0.9,NE) 12/18 (66.7) 12 (85.7) 3.4 (1.1, 12.4) 5/9 (55.6) 5 (83.3) 6.1 (4.4,NE) Gastro- 9/61 (14.8) 5 (71.4) 4.0 (0.8, NE) 71/145 (49.0) 62 (93.9)4.5 (3.5, 5.7) 54/114 (47.4) 44 (88.0) 4.9 intestinal (2.9, 7.6) Grade3-4 5/7 (71.4) 3 (50.0) NE (0.8, NE) 41/46 (89.1) 41 (97.6) 3.0 (1.9,4.3) 33/36 (91.7) 31 (88.6) 4.7 (1.9, 6.1) Endocrine 5/45 (11.1) 2(40.0) 24.3 (7.1, NE) 36/94 (38.3) 14 (41.2) NE (10.4, NE) 15/34 (44.1)4 (28.6) NE (0.71, NE) Grade 3-4 2/2 (100) 0 NE (NE, NE) 10/15 (66.7) 5(45.5) NE (4.5, NE) 7/7 (100.0) 3 (42.9) NE (0.43, NE) Hepatic 8/22(36.4) 6 (100.0) 7.0 (2.0, 27.1) 46/95 (48.4) 43 (95.6) 5.9 (3.3, 6.6)3/22 (13.6) 3 (100.0) 4.1 (4.0, 7.7) Grade 3-4 7/8 (87.5) 6 (100.0) 7.0(2.0, 27.1) 37/60 (61.7) 38 (100.0) 4.1 (3.0, 6.1) 2/5 (40.0) 2 (100.0)5.9 (4.0, 7.7) Pulmonary 4/5 (80.0) 4 (100.0) 3.3 (2.3, 9.1) 17/22(77.3) 16 (94.1) 6.1 (1.1, 8.3) 3/6 (50.0) 2 (66.7) 6.1 (6.0, 6.3) Grade3-4 1/1 (100) 1 (100.0) 2.3 (NE, NE) 2/3 (66.7) 2 (100.0) 4.2 (1.1, 7.3)1/1 (100) 1 (100.0) 4.7 (NE, NE) Renal 1/3 (33.3) 1 (100.0) 0.3 (NE, NE)4/17 (23.5) 3 (100.0) 1.7 (0.4, 3.7) 3/8 (37.5) 3 (100.0) 4.6 (0.6,16.1) Grade 3-4 0/1 0 — 3/6 (50.0) 3 (100.0) 1.7 (0.4, 3.6) 1/1 (100) 1(100.0) 4.6 (NE, NE)

The median progression-free survival reported for the combination ofnivolumab and ipilimumab in this study (11.7 months in BRAF-mutantpatients) is similar to that recently reported for the combination ofBRAF and MEK inhibition in BRAF-mutant metastatic melanoma (9.9 monthsfor vemurafenib and cobimetinib; 9.3 to 11.4 months for dabrafenib andtrametinib). Resistance to such targeted therapies is almost inevitablewhen used as monotherapy and in many cases is very rapid. The confirmedrate of objective response for combined nivolumab and ipilimumab (57.6%)is numerically higher than observed with PD-1 blockade alone in advancedmelanoma (nivolumab [40%] in treatment-naïve patients with wild-typeBRAF or pembrolizumab [37%] in ipilimumab-naïve patients).

The results of subgroup analyses suggest that the greatest benefit forthe combination of nivolumab and ipilimumab versus nivolumab alone mayoccur in the setting of negative PD-L1 tumor expression. In thePD-L1-positive group, both nivolumab alone and nivolumab plus ipilimumabresulted in a similar prolongation of progression-free survival ascompared with ipilimumab alone, although objective response rates werenumerically higher in the combination group versus either nivolumab oripilimumab alone. Thus, the use of PD-L1 as a biomarker may allowclinicians to make more informed decisions about the risk-benefit ofcombination therapy versus monotherapy. Nonetheless, the observation ofat least additive activity of the combination of ipilimumab andnivolumab in the setting of negative PD-L1 expression is of interest inmelanoma as well as in other tumor types in which PD-1 checkpointinhibitors are under evaluation.

The incidence of adverse events in this study was, in general, lowest inthe nivolumab group and highest in the combination group. The overallincidence of grade 3 or 4 drug-related adverse events was higher in thecombination group as compared with ipilimumab alone (39.6% versus18.6%), as a result of a slightly higher incidence in most adverseevents, particularly hepatic toxicity, where the rates of grade 3 or 4ALT/AST elevations were 6-8% for the combination and approximately 1%for ipilimumab alone. One drug-related death was reported in each of thenivolumab and ipilimumab groups but none in the combination group.Overall, the safety profile of the combination of nivolumab andipilimumab was consistent with previous experience with nivolumab oripilimumab alone. No new safety signals were identified, and adverseevents were manageable with established treatment guidelines as mostselect adverse events resolved with immune modulatory agents. These datasuggest that the combination of nivolumab and ipilimumab can be usedsafely in a broad range of clinical settings.

In summary, the report shows increased progression-free survival andobjective response rates for nivolumab alone and the combination ofnivolumab and ipilimumab, as compared with ipilimumab alone, inpreviously untreated advanced melanoma. Adverse events with thecombination were managed with established algorithms, with no studydrug-related deaths. Overall, nivolumab alone and the combination ofnivolumab and ipilimumab are promising treatment options for previouslyuntreated advanced melanoma.

SEQUENCES V_(H) amino acid sequence of 28-8 (Oryctolagus  cuniculus)(SEQ ID NO: 1) Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly Val Gln Cys Leu Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Thr Ile Thr Asn Tyr His Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Thr Ser Ser Gly Ile Gly Ser Ser Ser Thr Thr Tyr Tyr Ala Thr Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asn Leu Arg Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Tyr Phe Thr Asn Thr Tyr Tyr Ala Leu Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser SerV_(L) amino acid sequence of 28-8 (Oryctolagus  cuniculus)(SEQ ID NO: 2) Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Pro Gly Ala Arg Cys Ala Leu Val Met Thr Gln Thr Pro Ser Ser Thr Ser Thr Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Val Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Gln Arg Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Ser Ala Gly SerHeavy Chain CDR1 sequence of 28-8 (Oryctolagus  cuniculus)(SEQ ID NO: 3) Asn Tyr His Met PheHeavy Chain CDR2 sequence of 28-8 (Oryctolagus  cuniculus) (SEQ ID NO: 4) Val Ile Thr Ser Ser Gly Ile Gly Ser Ser Ser Thr Thr Tyr Tyr Ala Thr Trp Ala Lys GlyHeavy Chain CDR3 sequence of 28-8 (Oryctolagus  cuniculus) (SEQ ID NO: 5) Asp Tyr Phe Thr Asn Thr Tyr Tyr Ala Leu Asp IleLight Chain CDR1 sequence of 28-8 (Oryctolagus  cuniculus) (SEQ ID NO: 6) Gln Ala Ser Gln Ser Ile Ser Val Tyr Leu AlaLight Chain CDR2 sequence of 28-8 (Oryctolagus  cuniculus) (SEQ ID NO: 7) Ser Ala Ser Thr Leu Ala SerLight Chain CDR3 sequence of 28-8 (Oryctolagus  cuniculus) (SEQ ID NO: 8) Leu Gly Ser Ala Gly Ser Asp Asp Ala Ala

1. A method for treating a melanoma tumor in a patient in need thereofcomprising: (i) identifying a patient having a PD-L1 positive melanomatumor; and (ii) administering to the patient an anti-PD-1 antibody or anantigen-binding portion thereof that binds specifically to a human PD-1(“anti-PD1 antibody monotherapy”), wherein the patient is notadministered a combination of an anti-PD-1 antibody or anantigen-binding portion thereof and an anti-CTLA-4 antibody or anantigen-binding portion thereof that binds specifically to a humanCTLA-4 (“combination therapy”).
 2. (canceled)
 3. A method for treating amelanoma tumor in a patient in need thereof comprising administering tothe patient an anti-PD-1 antibody or an antigen-binding portion thereofthat binds specifically to a human PD-1 (“anti-PD-1 antibodymonotherapy”), wherein the patient is not administered a combination ofan anti-PD-1 antibody or an antigen-binding portion thereof and ananti-CTLA-4 antibody or an antigen-binding portion thereof that bindsspecifically to a human CTLA-4 (“combination therapy”), wherein thepatient is identified as having a PD-L1 positive melanoma tumor prior tothe administration. 4-8. (canceled)
 9. The method of claim 3, whereinthe patient is characterized by (i) extended progression-free survivalfor over 12 months following the administration, (ii) tumor sizereduction at least about 10% compared to the tumor size prior to theadministration, or (iii) both (i) and (ii). 10-13. (canceled)
 14. Amethod for selecting a patient suitable for an anti-PD-1 antibodymonotherapy comprising: (i) identifying a patient having a PD-L1positive melanoma tumor; and (ii) instructing a healthcare provider toadminister to the patient an anti-PD-1 antibody monotherapy.
 15. Themethod of claim 3, further comprising measuring a PD-L1 expression onthe melanoma tumor.
 16. The method of claim 3, wherein theadministration treats the melanoma tumor.
 17. The method of claim 15,wherein the measuring comprises providing a test tissue sample obtainedfrom the patient, the test tissue sample comprising tumor cells and/ortumor-infiltrating inflammatory cells.
 18. The method of claim 17,wherein the measuring further comprises assessing the proportion ofcells in the test tissue sample that express PD-L1 on the cell surface.19. The method of claim 18, wherein the test tissue sample is aformalin-fixed paraffin-embedded (FFPE) tissue sample.
 20. The method ofclaim 19, wherein the presence of PD-L1 is determined using an automatedIHC assay.
 21. The method of claim 20, wherein the IHC assay isperformed using an anti-PD-L1 monoclonal antibody that specificallybinds to the PD-L1 and wherein the anti-PD-L1 monoclonal antibody isselected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1, andany combination thereof.
 22. The method of claim 3, wherein the PD-L1positive melanoma tumor is characterized by having at least about 5% oftumor cells showing binding to the anti-PD-L1 antibody or anantigen-binding portion thereof.
 23. The method of claim 3, wherein theanti-PD-1 antibody or antigen-binding portion thereof cross-competeswith nivolumab for binding to human PD-1.
 24. The method of claim 3,wherein the anti-PD-1 antibody or antigen-binding portion thereof is achimeric, humanized or human monoclonal antibody or a portion thereof.25. The method of claim 3, wherein the anti-PD-1 antibody orantigen-binding portion thereof comprises a heavy chain constant regionwhich is of a human IgG1 or IgG4 isotype.
 26. The method of claim 3,wherein the anti-PD-1 antibody is nivolumab.
 27. (canceled)
 28. Themethod of claim 3, wherein the anti-PD-1 antibody or antigen-bindingportion thereof is administered at a dose ranging from 0.1 to 10.0 mg/kgbody weight once every 2, 3 or 4 weeks.
 29. The method of claim 28,wherein the anti-PD-1 antibody or antigen-binding portion thereof isadministered at a dose of 1 or 3 mg/kg body weight once every 3 weeks.30-48. (canceled)
 49. The method of claim 3, further comprisingadministering an anti-cancer agent.
 50. A kit for treating a patientafflicted with a melanoma tumor, the kit comprising: (a) a dosageranging from 0.1 to 10 mg/kg body weight of an anti-PD-1 antibody or anantigen-binding portion thereof; and (b) instructions for using theanti-PD-1 antibody or the antigen-binding portion thereof in the methodof claim
 3. 51. (canceled)